SOLVATION AND CONFORMATIONAL STABILITY OF PROTEINS AND by YUEN LAI SHEK

SOLVATION AND CONFORMATIONAL STABILITY OF PROTEINS AND DNA

by

YUEN LAI SHEK

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty Pharmacy, University of Toronto

© Copyright by Yuen Lai Shek 2016

ABSTRACT Solvation and Conformational Stability of Proteins and DNA Yuen Lai Shek Doctor of Philosophy 2016 Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University of Toronto

The work performed in this dissertation is devoted to understanding and quantifying solute-solvent interactions in the folded and unfolded states of proteins and DNA G-quadruplexes using volumetric techniques. We characterized the interactions of the protein stabilizer, glycine betaine (GB), with proteins and their functional groups through a combination of partial molar volume and adiabatic compressibility measurements of N-acetyl amino acid amides, oligoglycines, cytochrome c, ribonuclease A, lysozyme, and ovalbumin at GB concentrations ranging from 0 to 4 M. We evaluated the equilibrium (binding) constant, k, for the reaction in which a GB molecule binds each of the functionalities under study replacing four water molecules. We found that GB forms direct interactions with all protein groups studied here. In addition, the differential free energy of solute-solvent interactions in a concentrated GB solution and water, ΔΔGI, is negative for all the proteins studied and ΔΔGI becomes more favourable as the concentration of GB increases. We also employed volumetric measurements of lysozyme, apocytochrome c, ribonuclease A, ii

and α-chymotrypsinogen A in the solutions at 0 to 8 M urea to quantify the urea-induced solvation changes in the native and unfolded states of proteins. An increase in the concentration of urea to 8 M leads to a ~20% increase in the solvent accessible surface area of apocytochrome c. The urea-induced unfolding of ribonuclease A and α-chymotrypsinogen A is accompanied by increases in solvent accessible surface area of 1.9 ± 0.4 and 2.0 ± 0.6 times that of the native states, respectively. Furthermore, we characterized the volumetric properties of the folded and unfolded states of the Na+-stabilized antiparallel G-quadruplex conformation of Tel22, d[A(GGGTTA)3GGG], and the K+-stabilized hybrid-1 conformation of Tel26, d[AAAGGG(TTAGGG)3AA]. The coil-to-G-quadruplex transition of Tel26 accompanies a release of 434 ± 19 water molecules from its hydration shell to the bulk, which is more than four times the number of waters released compared to Tel22 (103 ± 44). We proposed that this extensive DNA dehydration originates from both the waters in direct contact with the domains that become buried in G-quadruplex formation and a general decrease in solute-solvent interactions all over the surface of the folded structure.

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ACKNOWLEDGEMENTS I would like to thank my research supervisor, Prof. Tigran V. Chalikian, for his encouragement, support and guidance throughout my thesis project. I would like to extend my gratitude to my advisory committee members: Prof. Robert B. Macgregor Jr., Prof. Régis Pomès, and Prof. Heiko Heeklotz, for their time, valuable input, and guidance. I also thank Prof. George Makhatadze for agreeing to serve as my external appraiser and Prof. Jeff Henderson for serving as my external examiner in my thesis defence. I want to express my gratitude to Dr. Soyoung Lee who introduced me to the Chalikian Laboratory and trained me with the numerous experimental techniques used for hydration studies. I would like to thank my laboratory colleagues and neighbours: Ikbae Son, Byul Gloria Kim, Nisha Patel, Dr. Eduardo Hidalgo Baltasar, Dr. Hiren Patel, Dr. Mozhgan Nazari, Dr. Rashid M. Abu-Ghazalah, Helen Y. Fan, Amir Amiri, Yang Li, Tameshwar Ganesh and Bita Zamiri for their stimulating discussion and assistance in the laboratory. I would also like to express my sincere appreciation to Dr. Mima Staikova who first introduced me to research in computational chemistry. Furthermore, I also want to thank my family and friends for their continuous support and encouragement throughout my graduate studies. Lastly, I would like to acknowledge the Canadian Institute of Health Research (CIHR) Protein Folding Training Program for financial support.

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TABLE OF CONTENTS ABSTRACT .............................................................................................................................. ii ACKNOWLEDGEMENTS ......................................................................................................iv TABLE OF CONTENTS ........................................................................................................... v PUBLISHED WORKS .............................................................................................................ix LIST OF TABLES ..................................................................................................................... x LIST OF FIGURES ................................................................................................................. xv CHAPTER 1: General Introduction ........................................................................................... 1 1.1: Proteins and DNA G-Quadruplexes Share Many Similarities........................................ 1 1.2 Equilibrium Dialysis Experiments and Preferential Interaction – An Early Approach to Investigate Protein-Osmolyte Interactions ............................................................................. 4 1.3 Solubility Studies and Tanford Transfer Model Free Energy of Protein Groups ............ 7 Table 1-1. Transfer Free Energies of Protein Functional Groups as a Function of Urea and Glycine Betaine Concentration, in cal mol-1 ...................................................................... 9 1.4 Vapor Pressure Osmometry and Local-Bulk Solute Partitioning Model Provide Valuable Insights into Osmolyte-Protein Interactions ........................................................................ 14 1.5 The Combination of Volumetric Measurements and the Solvent Exchange Model Is a Promising New Method for Studying Protein Solvation ..................................................... 19 1.6 Proposed Mechanisms of Urea-Induce Protein Denaturation ........................................ 23 1.7 Proposed Mechanisms of Glycine Betaine-Induce Protein Stabilization ...................... 24 1.8 Nucleic Acids Also Fold Into Globular Structures: DNA G-Quadruplex ..................... 27 1.9 DNA G-quadruplex-Cation Interaction Is Important for Stabilization .......................... 29 1.10 Hydration is a Major Determinant of DNA G-Quadruplex Stability .......................... 30 1.11: Partial Molar Volume ................................................................................................. 32 1.12: Partial Molar Adiabatic Compressibility .................................................................... 34 v

1.13: Outline of the Thesis ................................................................................................... 37 1.14: References ................................................................................................................... 40 CHAPTER 2: Volumetric Characterization of Interactions of Glycine Betaine with Protein Groups ...................................................................................................................................... 52 2.1 ABSTRACT ................................................................................................................... 53 2.2 INTRODUCTION ......................................................................................................... 54 2.3 MATERIALS AND METHODS ................................................................................... 55 2.4 RESULTS ...................................................................................................................... 58 2.5 DISCUSSION ................................................................................................................ 60 2.6 CONCLUDING REMARKS ......................................................................................... 89 2.7 ACKNOWLEDGEMENTS ........................................................................................... 90 2.8 REFERENCES .............................................................................................................. 91 CHAPTER 3: Glycine Betaine Interactions with Proteins: Insights from Volume and Compressibility Measurements ................................................................................................ 96 3.1 ABSTRACT ................................................................................................................... 97 3.2 INTRODUCTION ......................................................................................................... 98 3.3 MATERIALS AND METHODS ................................................................................. 100 3.4 RESULTS .................................................................................................................... 103 3.5 DISCUSSION .............................................................................................................. 116 3.6 CONCLUSION ............................................................................................................ 124 3.7 SUPPORTING INFORMATION ................................................................................ 125 3.8 FUNDING.................................................................................................................... 127 3.9 REFERENCES ............................................................................................................ 127 CHAPTER 4: Interactions of Urea with Native and Unfolded Proteins: A Volumetric Study ................................................................................................................................................ 133

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4.1 ABSTRACT ................................................................................................................. 134 4.2 INTRODUCTION ....................................................................................................... 135 4.3 MATERIALS AND METHODS ................................................................................. 137 4.4 RESULTS .................................................................................................................... 142 4.5 DISCUSSION .............................................................................................................. 146 4.6 CONCLUSION ............................................................................................................ 161 4.7 SUPPORTING INFORMATION ................................................................................ 162 4.8 ACKNOWLEDGEMENTS. ........................................................................................ 164 4.9 REFERENCES ............................................................................................................ 164 CHAPTER 5: Volumetric Characterization of Sodium-Induced G-quadruplex Formation Volumetric Characterization of Sodium-Induced G-quadruplex Formation ......................... 173 5.1 ABSTRACT ................................................................................................................. 174 5.2 INTRODUCTION ....................................................................................................... 175 5.3 MATERIALS AND METHODS ................................................................................. 177 5.4 RESULTS .................................................................................................................... 185 5.5 DISCUSSION .............................................................................................................. 200 5.6 CONCLUSIONS.......................................................................................................... 207 5.7 ACKNOWLEDGEMENTS ......................................................................................... 208 5.8 REFERENCES ............................................................................................................ 208 CHAPTER 6: Folding Thermodynamics of the Hybrid-1 Type Intramolecular Human Telomeric G-Quadruplex ....................................................................................................... 215 6.1 ABSTRACT ................................................................................................................. 216 6.2 INTRODUCTION ....................................................................................................... 217 6.3 RESULTS .................................................................................................................... 219 6.4 DISCUSSION .............................................................................................................. 232

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6.5 CONCLUDING REMARKS ....................................................................................... 241 6.6 MATERIALS AND METHODS ................................................................................. 242 6.7 ACKNOWLEDGEMENTS ......................................................................................... 248 6.8 REFERENCES ............................................................................................................ 248 CHAPTER 7: General Conclusion and Future Perspectives ................................................. 253 7.1 CONCLUSIONS.......................................................................................................... 253 7.2 FUTURE PROSPECT ................................................................................................. 258 Temperature Dependent Volumetric Measurements in Binary Solvents ...................... 258 Solvent Contribution Conformational Preferences ........................................................ 258 DNA G-quadruplex in binary solvents to probe hydration ............................................ 259 7.3 REFERENCES ............................................................................................................ 261 APPENDIX A: Urea Interactions with Protein Groups: A Volumetric Study ...................... 262 A.1 ABSTRACT ................................................................................................................ 263 A.2 INTRODUCTION ...................................................................................................... 264 A.3 MATERIALS AND METHODS ................................................................................ 267 A.4 RESULTS ................................................................................................................... 271 A.5 DISCUSSION ............................................................................................................. 274 A.6 CONCLUSIONS ......................................................................................................... 304 A.7 ACKNOWLEDGEMENTS ........................................................................................ 305 A.8 SUPPORTING INFORMATION ............................................................................... 306 A.9 REFERENCES............................................................................................................ 321

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PUBLISHED WORKS 1. Lee, S., Shek, Y.L., Chalikian, T.V. Urea interactions with protein groups: a volumetric study. Biopolymers. 93 (2010) 866-879. PMID: 20564051. (Appendix A) 2. Fan, H.Y.*, Shek, Y.L.*, Amiri, A., Dubins, D.N., Heerklotz, H., MacGregor, R.B., Chalikian, T.V. Volumetric characterization of sodium-induced G-quadruplex formation. J.Am.Chem.Soc. 133 (2011) 4518-4526. PMID: 21370889. *these authors contributed equally to this paper (Chapter 5) 3. Shek, Y.L., Chalikian, T.V. Volumetric characterization of interactions of glycine betaine with protein groups. J.Phys.Chem.B. 115 (2011) 11481-11489. PMID: 21866908. (Chapter 2) 4. Son, I., Shek, Y.L., Dubins, D.N., Chalikian, T.V. Volumetric characterization of tri-N-acetylglucosamine binding to lysozyme. Biochemistry. 51 (2012) 5784–5790. PMID: 22732010 5. Orava, E.W., Jarvik, N., Shek, Y.L., Sidhu, S.S., Gariepy, J. A short DNA aptamer that recognizes TNFα and blocks its activity in vitro. ACS Chem Biol. 8 (2013) 170-178. PMID: 23046187 6. Shek, Y.L., Chalikian, T.V. Interactions of glycine betaine with proteins: insights from volume and compressibility measurements. Biochemistry. 52 (2013) 672-680. PMID: 23293944 (Chapter 3) 7. Kim, B. G., Shek, Y. L., Chalikian, T. V. Polyelectrolyte effects in G-quadruplex. Biophys. Chem. 184 (2013) 95-100. 8. Son, I., Shek, Y. L., Dubins, D. N., Chalikian, T. V. Hydration changes accompanying helix-to-coil DNA transitions. J. Am. Chem. Soc. 136 (2014) 4040-4047. 9. Shek, Y.L.*, Noudeh, G.D.*, Nazari, M., Heerklotz, H., Abu-Ghazalah, R.M., Dubins, D., Chalikian, T.V. Folding thermodynamics of the hybrid-1 type intramolecular human telomeric G-quadruplex. Biopolymers. 101 (2014) 216-227. *these authors contributed equally to this paper (Chapter 6) 10. Son, I.*, Shek, Y. L.*, Tikhomirova, A., Baltasar, E. H., Chalikian, T. V. Interactions of urea with native and unfolded proteins: a volumetric study. J. Phys. Chem. B. 118 (2014) 13554-13563. *these authors contributed equally to this paper (Chapter 4)

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LIST OF TABLES Chapter 1

Table 1-1

Transfer Free Energies of Protein Functional Groups as a Function of Urea and Glycine Betaine Concentration, in cal/mol.

Chapter 2

Table 2-1

Density of Solvent, 0, Coefficient of Adiabatic Compressibility of Solvent, S0, Excess Partial Molar Volume of Water, V°1, and Excess Partial Molar Adiabatic Compressibility of Water, K°S1, as a Function of GB Concentration

Table 2-2

Relative Molar Sound Velocity Increments, [U] (cm3 mol-1), of Solutes as a Function of GB Concentration

Table 2-3

Partial Molar Volumes, V° (cm3 mol-1), of Solutes as a Function of GB Concentration

Table 2-4

Partial Molar Adiabatic Compressibilities, K°S (10-4 cm3 mol-1 bar-1), of Solutes as a Function of GB Concentration

Table 2-5

Partial Molar Volume Contributions of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), V (cm3 mol-1), as a Function of GB Concentration

Table 2-6

Partial Molar Adiabatic Compressibility Contributions of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), KS (10-4 cm3 mol-1bar-1), as a Function of GB Concentration

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Table 2-7

The Correction Factor, 1, the Number of Binding Sites for Water, n, Equilibrium Constants, k, and Changes in Volume, V0, and Adiabatic Compressibility, KS0, Accompanying the Binding of GB to Amino Acid Side Chains and the Glycyl Unit in an Ideal Solution

Table 2-8

Change in Free Energy of Cavity Formation, GC (kcal mol-1), of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), Calculated as a Function of GB Concentration Using Eq. (6)

Table 2-9

Differential Free Energy of Interaction, GI (kcal mol-1), of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), Calculated as a Function of GB Concentration Using Eq. (8)

Table 2-10

Changes in Free Energies, Gtr(exp) (kcal mol-1), Calculated for the Water-to-GB Transfer of Amino Acid Side Chains and the Glycyl Unit (-CH2CONH-) Using Eq. (1)

Table 2-11

Calculated, Gtr(calc) (cal mol-1), and Experimental, Gtr(exp) (cal mol-1), Free Energies for the Transfer of the Amino Acid Side Chains and the Glycyl Unit (-CH2CONH-) from Water to 1 M GB

Chapter 3

Table 3-1

The Partial Molar Volumes of Proteins, V° (cm3 mol-1), of the Proteins at Various GB Concentrations

Table 3-2

The Partial Molar Adiabatic Compressibilities, K S ° (10-4 cm3 mol-1 bar-1), of the Proteins at Various GB Concentrations

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Table 3-3

Protein Solvent Accessible Surface Areas, SA (Å2), Correction 0, Changes in Volume, V0 (cm3 mol-1), and Adiabatic Compressibility, KS0 (10-4 cm3 mol-1 bar-1), for GB-Protein Association in an Ideal Solution, and Equilibrium Constants Determined from Volume, k(vol) (M-1) and Compressibility, k(comp) (M-1), Data

Table 3-4

Changes in Volume, V0 (cm3 mol-1), and Adiabatic Compressibility, KS0 (10-4 cm3 mol-1 bar-1), for GB-Protein Association in an Ideal Solution Estimated Based on Low Molecular Weight model Compound Data

Table 3-S1

The Extinction Coefficients of the Proteins,  (M-1 cm-1), at Various GB Concentrations

Table 3-S2

The Relative Molar Sound Velocities, [U] (cm3 mol-1), of the Proteins at Various GB Concentrations

Chapter 4

Table 4-1

Extinction Coefficients (M-1 cm-1) of the Proteins as a Function of Urea

Chapter 5

Table 5-1

Coil-to-Quadruplex Transition Temperatures, TM, Volumes, Vtr, and Expansibilities, Etr, Determined from Pressure Perturbation Calorimetric (PPC), Vibrating Tube Densimetry (VTD), and High Pressure Measurements (HP) at Different NaCl Concentrations

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Table 5-2

Molecular Volumes, VM, and Solvent Accessible Surface Areas, SA, of the ODN in the Coil and Quadruplex Conformations

Chapter 6

Table 6-1

G-Quadruplex-to-Coil Transition Temperatures, TM, and van't Hoff, HvH, and Calorimetric, Hcal, Enthalpies Determined from UV Melting and DSC Measurements

Table 6-2

Molecular, VM, and van der Waals, VW, Volumes and Solvent Accessible Surface Areas, SA, of the ODN in the Coil and Quadruplex Conformations

Appendix A

Table A-1

Partial Molar Volume Contributions of Amino Acid Side Chains, V (-R) (cm3 mol-1), as a Function of Urea Concentration

Table A-2

Partial Molar Adiabatic Compressibility Contributions of Amino Acid Side Chains, KS (-R) (10-4 cm3 mol-1bar-1), as a Function of Urea Concentration

Table A-3

The Correction Factor, 1, the Number of Binding Sites for Water, n, Equilibrium Constants, k, and Changes in Volume, V0, and Adiabatic Compressibility, KS0, Accompanying the Binding of Urea to Amino Acid Side Chains and the Glycyl Unit in an Ideal Solution

Table A-S1

Relative Molar Sound Velocity Increments, [U] (cm3 mol-1), as a Function of Urea Concentration

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Table A-S2

Partial Molar Volumes, V° (cm3 mol-1), as a Function of Urea Concentration

Table A-S3

Partial Molar Adiabatic Compressibilities, K°S (10-4 cm3 mol-1 bar-1), as a Function of Urea Concentration

Table A-S4a

Dissociation Constants, pKa, and Changes in Volume, V, and Adiabatic Compressibility, KS, Accompanying Protonation of Titrable Side Chains at 0 M Ureaa

Table A-S4b

Dissociation Constants, pKa, and Changes in Volume, V, and Adiabatic Compressibility, KS, Accompanying Protonation of Titrable Side Chains at 2 M Urea

Table A-S4c

Dissociation Constants, pKa, and Changes in Volume, V, and Adiabatic Compressibility, KS, Accompanying Protonation of Titrable Side Chains at 4 M Urea

Table A-S4d

Dissociation Constants, pKa, and Changes in Volume, V, and Adiabatic Compressibility, KS, Accompanying Protonation of Titrable Side Chains at 6 M Urea

Table A-S4e

Dissociation Constants, pKa, and Changes in Volume, V, and Adiabatic Compressibility, KS, Accompanying Protonation of Titrable Side Chains at 8 M Urea

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LIST OF FIGURES Chapter 1

Figure 1-1

Common protein denaturants: (a) urea, (b) guanidine hydrochloride, and protein stabilizers: (c) glycine betaine, (d) trimethylamine N-oxide (TMAO).

Figure 1-2

Hoogsteen interaction in a G-quartet.

Chapter 2

Figure 2-1

The partial molar volume (panel A) and adiabatic compressibility (panel B) contributions of the leucine side chain as a function of GB activity. The experimental points were fitted using Eqs. (4) (panel A) and (5) (panel B).

Figure 2-2

Water-to-GB transfer free energies of the amino acid side chains and the glycyl unit plotted as a function of GB activity.

Chapter 3

Figure 3-1

The partial molar volumes of cytochrome c (a), ribonuclease A (b), lysozyme (c), and ovalbumin (d) as a function of GB activity.

Figure 3-2

The partial molar adiabatic compressibilities of cytochrome c (a), ribonuclease A (b), lysozyme (c), and ovalbumin (d) as a function of GB activity.

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Figure 3-3

The free energies of direct GB-protein interactions for cytochrome c (black), ribonuclease A (red), lysozyme (green), and ovalbumin (blue) as a function of GB activity.

Chapter 4

Figure 4-1

Far-UV CD spectra of apocytochrome c at different urea concentrations.

Figure 4-2

Urea dependences of the molar ellipticities at 219 nm for ribonuclease A (a) and -chymotrypsinogen A (b). Experimental data were fitted by Eq. (3) (solid lines).

Figure 4-3

Urea dependences of the partial molar volumes of lysozyme (a), apocytochrome c (b), ribonuclease A (c), and -chymotrypsinogen A (d). Experimental data in panels A and B were fitted by Eq. (1), while those in panels C and D were fitted by Eq. (3) (solid lines). The pre- and post-denaturational baselines of the protein denaturation profiles in panels C and D were fitted by Eq. (1) (dashed red lines).

Figure 4-4

Urea dependences of the partial molar adiabatic compressibilities of lysozyme (a), apocytochrome c (b), ribonuclease A (c), and -chymotrypsinogen A (d). Experimental data in panels A and B were fitted by Eq. (2), while those in panels C and D were fitted by Eq. (3) (solid lines). The pre- and post-denaturational baselines of the protein denaturation profiles in panels C and D were fitted by Eq. (2) (dashed red lines).

Figure 4-S1

Near- (panel a) and far-UV (panel b) CD spectra of lysozyme at different urea concentrations.

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Figure 4-S2

Near-UV CD spectrum of apocytochrome c in the absence of urea.

Chapter 5

Figure 5-1

(a) Circular dichroism spectra of the ODN at 25 °C in the presence and absence of NaCl. The DNA was initially in a non-folding buffer consisting of 10 mM tetrabutylammonium phosphate titrated to pH 7.0, 1 mM EDTA, and 0.1 mM NaN3. The NaCl concentration (in mM) for each spectrum is given in the inset. (b) The dependence of the molar ellipticity of the ODN at 295 nm on the NaCl concentration. Experimental data were approximated with Eq. (5) (solid line).

Figure 5-2

Temperature dependent changes in the partial molar volume of the ODN at 20 mM (panel A) and 50 mM (panel B) NaCl. Temperature was varied from high to low. Experimental data were fitted by the two-state model of thermal denaturation.

Figure 5-3

Changes in the partial molar volume (panel A) and adiabatic compressibility (panel B) of the ODN plotted against the NaCl concentration at 25 °C. The DNA was initially in a non-folding buffer consisting of 10 mM tetrabutylammonium phosphate titrated to pH 7.0, 1 mM EDTA, and 0.1 mM NaN3. Experimental data were approximated with Eq. (5) (solid lines).

Figure 5-4

Pressure dependence of the helix-coil transition temperature, TM, as a function of hydrostatic pressure at 20 mM (triangles), 50 mM (circles), and 100 mM (squares) NaCl. The lines are linear least-square fits of the data; 0.1 MPa is atmospheric pressure.

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Figure 5-5

PPC-determined temperature dependences of the apparent molar expansibility of the ODN at 50 and 100 mM NaCl. The value of ΔV is obtained as illustrated by integrating the peak from a progress baseline; the difference between extrapolated pre- and post-transition baselines yields the expansibility change, E. The position of maximum of the peak corresponds to the T M.

Figure 5-6

The dependence of the transition volume on the temperature for the three methods employed in this work. The slope of the temperature dependence equals the expansibility change for the reaction. A linear, least-squares fit of the data yields a slope of −0.87 ± 0.16 cm3 mol-1 K-1. The experimental method employed to obtain a particular datum is given in the inset.

Chapter 6

Figure 6-1

(a) CD spectra of the ODN taken in a non-folding buffer in the absence of K+ ions () and after 0 (), 30 (), 60 (), and 540 () min following addition of 30 mM KCl. Inset: time dependence of the molar ellipticity at 242 nm. (b) Time dependence of the molar ellipticity at 268 nm following addition of 30 mM KCl. Experimental data were approximated by an exponential function (solid line).

Figure 6-2

KCl dependences of the molar ellipticities of the ODN at 268 (panel A) and 295 (panel B) nm. Experimental data were approximated with Eq. (1) (solid line).

Figure 6-3

Salt-dependence of the melting temperature.

Figure 6-4

DSC thermogram of the ODN at 75 mM KCl. The data have been fitted (red solid line) based on the two-state approximation of thermal denaturation.

xviii

Figure 6-5

PPC melting profile of the ODN at 75 mM KCl. The data have been fitted (solid line) based on the two-state approximation as described previously of thermal denaturation.74, 75 The dashed red line is the temperature-dependent change in baseline.

Figure 6-6

KCl dependence of the change in volume, V, of the ODN at 25 °C. Experimental data were approximated with Eq. (1) (solid line).

Figure 6-7

KCl dependence of the change in relative molar sound velocity increment, [U], of the ODN at 25 °C. Experimental data were approximated with Eq. (1) (solid line).

Figure 6-8

Transition volumes, V, of the ODN plotted as a function of temperature.

Appendix A

Figure A-1

The volume (panel A) and compressibility (panel B) contributions of the leucine side chain as a function of urea. The fitting of the experimental data (continuous lines) was accomplished using Eq. (8) (panel A) and Eq. (10) (panel B) as explained in the text.

xix

Figure A-2

The differential free energy of solute-solvent interactions, GI, in a urea solution and water calculated as a function of urea concentration with Eq. (12); plot 1 – k = 0.04 M (Ser, Asp); plot 2 – k = 0.06 M (Glu); plot 3 – k = 0.08 M (Gly, Ile); plot 4 – k = 0.09 M (Gln); plot 5 – k = 0.11 M (Asn, His); plot 6 – k = 0.12 M (Arg); plot 7 – k = 0.14 M (Thr, Trp); plot 8 – k = 0.16 M (glycyl backbone); plot 9 – k = 0.18 M (Cys); plot 10 – k = 0.19 M (Phe); plot 11 – k = 0.21 M (Ala); plot 12 – k = 0.22 M (Val, Leu); plot 13 – k = 0.23 M (Lys); plot 14 – k = 0.26 M (Tyr); plot 15 – k = 0.31 M (Met); plot 16 – k = 0.39 M (Pro). For the alanine, phenylalanine, tryptophan side chains and the glycyl unit, the average of the two binding constants, k, presented in Table A-3 was used in the calculations.

Figure A-3

The differential free energy of solute-solvent interactions, GI, in a 2 M urea solution and water calculated for the amino acid side chains and the glycyl unit (BB) from water to 2 M urea. For the alanine, phenylalanine, tryptophan side chains and the glycyl unit, the average of the two binding constants, k, presented in Table A-3 was used in the calculations.

Figure A-4

The differential free energy of solute-solvent interactions, GI, in a urea solution and water for a solute with five binding sites calculated as a function of urea concentration with Eq. (14). The urea binding constant, k, used in calculations is 0.15 M. The concentrations of a solute are 0.1 M (red), 1 M (blue), and 3 M (green).

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CHAPTER 1: General Introduction 1.1: Proteins and DNA G-Quadruplexes Share Many Similarities When a water-soluble organic cosolvent is added to a protein or nucleic acid that is dissolved in water as the principal solvent, the structure of the protein or nucleic acid may be stabilized, destabilized, or may exhibit no change. A cosolvent may be classified as stabilizing or destabilizing depending on its effect on the conformational stability of biopolymers. Some cosolvents have been named osmolytes for their ability to regulate the osmolality in the presence of cellular stresses due to extreme salinity, pressure, or temperature in the living environment (1–4). In protein studies, a cosolvent that exerts a stabilizing effect on the folded conformation of proteins is known as a stabilizing osmolyte, while a cosolvent that causes protein denaturation is defined as a destabilizing osmolyte. Osmolytes are chemically diverse and they may fall in the classes of methylamines, polyols, or amino acids (3). Numerous studies investigating the effects of osmolytes on protein stability have been reported (3,5–10). In the current dissertation, the terms osmolyte and cosolvent are used interchangeably. The strongest protein stabilizers are trimethylamine N-oxide (TMAO) and glycine betaine (1). The most effective destabilizing osmolytes are urea and guanidine hydrochloride, which are commonly used as protein denaturants (5). (a)

(b)

(c)

(d)

Figure 1-1. Common protein denaturants: (a) urea, (b) guanidine hydrochloride, and protein stabilizers: (c) glycine betaine, (d) trimethylamine N-oxide (TMAO). In fact, urea is known not only for its ability to denature proteins, but also its role as a denaturant for nucleic acids. Studies have shown that urea lowers the thermal stability of secondary and tertiary structures of nucleic acids (11–13). There are, however, exceptions to this general observation. For example, the tetramolecular G-quadruplex of [d(TTGGGGTT)]4 is resistant to denaturation even at 7 M urea (14). Thermal denaturation of the Sr2+-stabilized G-quadruplex of thrombine binding

1

aptamer (TBA), d[G2T2G2TGTG2T2G2], demonstrated that urea, even up to 7 M, exerts a relatively insignificant destabilizing effect of 2-3°C, whereas a quadruplex formed by the same oligonucleotide but stabilized by K+ undergoes much greater destabilization (at 5 M urea, ΔTm = -7.4 °C). The effect of stabilizing osmolytes on the conformational stability of nucleic acids is even more complex. Not all nucleic acid conformations respond equally to stabilizing osmolytes. Glycine betaine has been reported to destabilize duplex DNA but stabilize the G-quadruplex conformation (15). Hong and coworkers propose that urea acts as an effective nonspecific nucleic acid denaturant via preferential interactions with the polar amide-like surface groups of guanine, cytosine and thymine exposed upon the unfolding of duplex DNA (16). They propose that glycine betaine destabilizes duplex DNA because of its preferential interactions with cytosine and guanine exposed in the unfolded state (16). Nevertheless, no conclusive explanation has been proposed for glycine betaine‘s action on DNA G-quadruplexes (15). The energetics governing both protein folding and nucleic acid folding can be rationalized in terms of the differential energetics of solute-solvent and intra-solute interactions. It is therefore of paramount importance to study the solute-solvent interactions of proteins and nucleic acids to understand the forces governing their folding/unfolding events. Volumetric techniques offer a powerful approach to studying solute-solvent interactions (17). Volumetric measurements such as partial molar volume and adiabatic compressibility are sensitive to the entire spectrum of changes in solvation of solvent-exposed functional groups of proteins and nucleic acids (18). These measurements can detect changes in solute-solvent interactions associated with folding/unfolding and molecular binding events. Volumetric techniques have been employed extensively to characterize the hydration of proteins and nucleic acids and their functional groups (18). Recently, the Chalikian laboratory reported a series of studies that employed a combination of volumetric measurements to characterize the interactions of protein functional groups with water and urea to quantify the contributions of amino acid side chains and the glycyl unit to the stability of the folded and unfolded states of globular proteins (7,19). Although globular proteins and DNA G-quadruplexes belong to different classes of biopolymers, nonetheless, the geometric shapes of both biopolymers are 2

contingent upon the water molecules solvating these biomolecules (17,20,21). These two classes of biopolymers share many similar characteristics. Specifically, globular proteins and DNA G-quadruplexes share the following common features: (1)

Both globular proteins and DNA G-quadruplexes exhibit tertiary structures with a globular shape (22).

(2)

The conformational preferences of both globular proteins and DNA G-quadruplexes depend on the composition of the solvent medium and the type cosolvent(s) present.

(3)

Both globular proteins and DNA G-quadruplexes have internal voids that render them pressure-sensitive.

(4)

Both globular proteins and DNA G-quadruplexes are capable of folding/unfolding cooperatively in a two-state manner and this property allows for the analysis of their conformational transitions induced by adjusting the solvent composition, temperature or pressure.

(5)

Hydration (solute-solvent interactions) is a major driving force that regulates the conformational preferences of both proteins and nucleic acids (17,20,21).

In recognition of these similarities, parallel studies of the conformational stability of proteins and DNA G-quadruplex can produce unique insights. The objective of my dissertation is to characterize solute-solvent interactions in the folded and unfolded states of proteins and DNA G-quadruplexes using volumetric techniques. To fully understand the solute-solvent interactions involved in determining the conformational stability of proteins and nucleic acids, the investigation must be conducted in a sequential manner. First, the role of water in the conformation preferences of proteins and nucleic acids must be extensively studied and the associated thermodynamic parameters must be determined. Second, proteins and nucleic acids should be studied in the presence of cosolvents to investigate the role of solute-solvent interactions in the conformational stability of proteins and DNA in a mixed-solvent system versus in water alone. For nearly a century, hydration has been extensively investigated in globular proteins (19,23–27); however, to date, no more than twenty-five hydration studies of DNA G-quadruplexes have been reported in the literature (21,28–39). For this reason, we have chosen to investigate hydration of DNA 3

G-quadruplexes stabilized by Na+ and K+ and to study the solvation of globular proteins in aqueous solutions of urea and glycine betaine. Studying the solvation of proteins and DNA G-quadruplex will help clarify the group-specific solute-solvent interactions that collectively determine the overall stability and conformational preferences of these biomolecules.

1.2 Equilibrium Dialysis Experiments and Preferential Interaction – An Early Approach to Investigate Protein-Osmolyte Interactions An approach pioneered by Timasheff for studying the interactions between proteins and osmolytes is based on measuring the preferential interaction parameter, (∂µ2/∂m3)T,P,µ ,m = (∂µ3/∂m2)T,P,µ ,m , which reflects the mutual perturbation of the 1

2

1

3

chemical potentials of protein and cosolvent when the protein is added to the binary solvent. As a consequence, the composition of the solvent in the immediate vicinity of the protein differs from that in the bulk. The free energy of transferring the protein from water to an osmolyte solution, Δµtr, is given by (40):

   tr    2  dm3 m3 T , P ,  ,m 0 1 2 m3

[1]

The application of the cyclic rule to the definition of the preferential interaction parameter, (∂µ3/∂m2)T,P,µ ,m , yields the preferential binding parameter, Г23, (41): 1

 m  23   3   m2 T , P ,1 ,3

3

  2    3          m2 T , P ,1 ,m3  m3 T , P ,1 ,m2     2    3    3    3 T , P ,1 ,m2      m3 T , P ,1 ,m2  m3 T , P ,1 ,m2

[2]

The preferential binding parameter, Г23, is a measure of the excess of osmolyte in the immediate vicinity of the protein relative to the bulk phase (40). A positive preferential binding parameter, Г23, signifies an accumulation of osmolyte near the protein relative to the bulk, while a negative Г23 indicates an exclusion of osmolytes from the vicinity of the protein. Contrary to a common misconception, the term ―immediate vicinity‖ does not imply a physically defined volume or compartment around the protein, but instead, it refers to the domain in which the protein exercises a thermodynamic influence on the 4

osmolyte and water molecules. In this domain, the water and osmolyte molecules are chemically identical to their counterparts in the bulk; however, the local density and composition of the solvent components are different from those in the bulk solvent (41). Symmetrically, the preferential hydration parameter, Г21, reflects the excess of water in vicinity of the protein. Application of the Gibbs-Duhem formula to Eq. (2) relates the preferential binding parameter, Г23, to the preferential hydration parameter, Г21,

 m  m  m  m 21   1    1  3    1 23 m3  m2 T , P ,  ,  m3  m2 T , P , 1 , 3 1 3

[3]

where subscripts i = 1,2 and 3 denote water, protein, and osmolyte, respectively; T is temperature; P is pressure; µi is the chemical potential of component i; and mi is the molal concentration of component i (42). Experimentally, preferential binding parameter, Г23, is determined from equilibrium dialysis and high precision densimetry measurements:

 m  M  (v o  v  o ) M  g  23   3   2  3   2 o 2 2 M 3 (1   o v3 )  m2 T , P ,1 ,3 M 3  g 2 T , P ,1 ,3

[4]

where gi, is the specific concentration of component i expressed in grams of component i per gram of water; Mi is the molecular weight of component i; ρo is the density of the solvent; v3 is the partial specific volume of component 3 (cosolvent); v2o is the apparent specific volume of component 2 (protein) at equal molalities of solvent components in the protein solution and the reference solvent; and v2,o is the apparent specific volume of component 2 at equal chemical potentials of the solvent components in the protein solution and the reference solvent (41). Operationally, the preferential binding parameter, Г23, is described by the following relationship:

 m  minside  m3outside 23   3   3 m2  m2 T , P , 1 , 3

[5]

5

where m2 is the molal concentration of component 2 inside the dialysis bag; m3inside and m3outside are the molalities of component 3 inside and outside the dialysis bag, respectively (43).

Timasheff and coworkers have determined the preferential binding parameter of many water soluble proteins in various stabilizing and destabilizing osmolytes (44–48). All proteins, with the exception of ribonuclease A, exhibit positive preferential binding parameters, Г23, with respect to denaturing cosolvents (44). In 6 M guanidine hydrochloride, ribonuclease A exhibits a preferential binding parameter, Г23, close to 0 (44). In a study examining the interactions between urea and nine model proteins in their denatured states, Timasheff and coworkers found that Г23 ranges between 2 to 96 mol/mol in 8M urea at pH 4.0 (45). More recently, Xie and Timasheff observed that at 20°C, lysozyme remains native at pH 7.0 up to 9.5 M urea, while it exhibits a urea-induced denaturation at pH 2.0 (46). This observation allowed them to employ lysozyme at pH 7.0 as a model to investigate the interactions of urea with native proteins (46). The sign of Г23 of lysozyme in urea is positive at 1.5 M urea and above at both pH 2.0 and 7.0 (46). According to Eq. (3), a positive sign in preferential binding parameter translates into preferential exclusion of water. This does not signify that there is no water in the immediate vicinity of the protein, but rather, it suggests that the affinity of protein for the denaturants is greater than that for water molecules (46). In contrast to denaturants, the sign of the preferential binding parameters of proteins in solutions of stabilizing osmolytes is negative. For example, the preferential binding parameter of lysozyme decreases from -6.45 to -13.5 mol/mol as the concentration of glycine betaine increases from 0.7 M to 2.0 M, while the preferential binding parameter of bovine serum albumin (BSA) decreases from -26.9 to -72.5 mol/mol over the same range of glycine betaine concentrations (47). These results indicate that stabilizing osmolytes are preferentially excluded from the protein domain (48). Although equilibrium dialysis experiments offer valuable insights into the distribution of water and osmolyte molecules around proteins, preferential interaction and binding parameters describe the differential interactions of proteins with water and 6

cosolvents over the entire protein surface. These parameters do not specify the locality of protein-cosolvent interactions. Furthermore, the technique is limited to proteins as it cannot be applied to studying low molecular weight model compounds mimicking protein groups. As such, this technique cannot be used for investigating specific interactions between individual protein groups and osmolytes.

1.3 Solubility Studies and Tanford Transfer Model Free Energy of Protein Groups Originated in the 1930s, solubility studies of low molecular model compounds mimicking protein groups have been designed to determine the free energy associated with the transfer of a protein group from water to a binary solvent containing osmolyte and water, Δµtr (49,50). In these studies, the solubility limit of low molecular weight model compounds in water, s21, and in the binary solvent, s23, are measured. The free energy of transferring a solute from water to osmolyte solution, Δµtr, is the difference in the chemical potential of solute in water, µ21o, and in the binary solvent, µ23 o, given by a   s  s    o o  tr   23   21  RT ln 21   RT ln 21 21   RT ln 21   RT ln 21   a23    23 s23   s 23    23 

[6]

where R is the universal gas constant; T is the temperature; and a21   21s21 and a23   23 s23 are the activities of the solute in water and water-cosolvent mixture,

respectively. Since the activity coefficients of the solute in water, γ21, and in the binary solvent, γ23, are, generally, not known, it is conventionally assumed that γ21/γ23 ≈ 1 (51–54). Under this assumption, the apparent transfer free energy is given by: s   tr '  RT ln 21   s23 

[7]

The laboratories of Tanford and Bolen groups determined the apparent transfer free energies for amino acid side chains, Δµtr‘, using an additive scheme in which the Δµtr‘ of glycine is subtracted from the Δµtr‘ of the amino acid of interest (51–53). More recently, Venkatesu and coworkers have employed the additive scheme on cyclic dipeptides, instead of zwitterionic amino acids, to determine the apparent transfer free 7

energy of amino acid side chains, Δµtr‘ (55). The Δµtr‘ for the peptide backbone has been calculated using the additive scheme on various model compounds including oligoglycines, and diketopiperazine (51,54,56–58). The Δµtr‘ for amino acid side chains and peptide backbone in urea and glycine betaine solutions are summarized in Table 1-1.

8

Table 1-1. Transfer Free Energies of Protein Functional Groups as a Function of Urea and Glycine Betaine Concentration, in cal mol-1 Protein Group

1 M urea a

Trp Phe

-141.46 , b -101.19 a -83.11 , b -42.84 a

Leu

-54.57 , -14.30 b

 2 M urea -269.68 , d -270 c -169.09 , d -180 -111.63 c, d -110 z -103±21

a

Ile Val Met

-38.43 , 1.84 b a -21.65 , b 18.62 a -48.34 , b -8.07 a

Ala

His Thr Ser Tyr LysHCl ArgHCl NaGlu NaAsp Gln Asn Pro Gly

Glycyl

d

6 M urea

d

8 M urea

d

1 M Betaine

-505

-730

-920

-369.93

e

-330

-470

-600

-112.93

e

-155

-225 -221±22 z

-295 -296±22 z

c

-76.66

-225

-325

-50.51 a, b -10.24 a -22.09 , b 18.18 -20.56 a, 19.71 b -45.08 a, -4.81 b a -22.76 , 17.51 b -21.17 a, 19.10 b 0.62 a, 40.89 b 3.55 a, 43.82 b -54.81 a, -14.54 b -38.79 a, 1.48 b -17.65 a, 22.62 b

-100.83 c, d -100 c -43.43 , d -40

0a

0c

15

10

10

-160

-205

-255

-35.97

-60

-90

-115

0.33

f

g

229.08 , h 56.77 , 142.93 i

g

64.08 , h 55.97 , 60.03i

e

e

-395

-580

-213.09 e

-735

-45.32 c

-171.99 e

-42.14 c

-109.45 e

1.69 c

-112.08 e

7.35c

-116.56 e c

-130

-190

-230

7.57 e

-225

-330

-430

33.17 e

-35.34 c

-69.15 n, -10 p, -145 q, -18.13 r, -147.37 s, -79.48 t, -82.75 u, -113.42 v, -81.66 w -6±3 x -160±12y

117.88

-41.85 e

c

-94.29 , -80 d -103.02 c, -135 d

f

e

4.77 e, g -41.29 , h 59.01 , i 8.86

-40.05 c -89.94 , -225 d

4 M Betaine

-14.16 e

-415

c

-7.67 , d 0

79.61

3 M Betaine

-19.63 e

c

-102.36 , d -115

2 M Betaine

e

-17.73 , 62.05 f -1.27

-43.12 c

-4.69 , b 0.63

-39 m, -14.79 r, -71.82 s, -65.70 t, -43.30 u, -68.76 v, -50.77 w

4 M urea

c

-125.16 e 0 -20 p, -205 q, -31.84 r, -225.60 s, -127.11 t, -128.72 u, -176.36 v, -128.18 w

0 -96 o -35 p, -305 q, -46.36 r, -313.89 s, -179.83 t, -180.13 u, -246.87 v, -180.03 w -21±4 x -342±12y

0

0

0

67 m, 60.9 l, 34.27 r, -37.06 s, 43.45 t, -1.39 u, 3.15 v, 13.52 w

97.99 l, 21.84 r, -40.93 s, -37.82 t, -9.54 u, -39.37 v, -18.96 w

0

p

-60 , -310 q, -47.58 r, -411.92 s, -225.98 t, -229.75 u, -318.95 v, -228.50 w -57±4 x -337±12y

9

52.72 r, -129.16 s, -84.00 t, -38.22 u, -106.58 v, -53.48 w

203.58 l, 103.82 r, -218.95 s, -177.78 t, -57.56 u, -174.44 v, -97.63 w

‘

a

Apparent free energy of transfer to 1 M urea determined using zwitterionic amino acids, calculated by dividing the 2 M urea Δµtr values by 2, from Ref(52).

b

Glycine activity coefficient corrected free energy of transfer to 1 M urea determined using zwitterionic amino acids, calculated by dividing the 2 M values by 2, from Ref(53).

c

Apparent free energy of transfer to 2 M urea determined using zwitterionic amino acids, from Ref(57).

d

Apparent free energy of transfer to 2 M, 4 M, 6 M and 8 M urea determined using zwitterionic amino acids, from Ref(51).

e

Apparent free energy of transfer to 1 M betaine measured using zwitterionic amino acids, from Ref(52).

f

Apparent free energy of transfer to 1 M, 2 M and 4 M betaine measured using cyclic dipeptides c(LA)-c(AG), from Ref(55).

g

Calculated by c(AG) – c(GG) , from Ref(55).

h

Calculated by c(AA) – c(AG) , from Ref(55).

i

Calculated by [c(AA) – c(GG)]/2, from Ref(55).

l

Calculated by c(GG)/2, from Ref(55).

m

Calculated by c(GG)/2, from Ref(58).

n

Calculated by c(GG)/2, from Ref(57).

o

Calculated by c(GG)/2, from Ref(56).

p

Calculated by diglycine – glycine, from Ref(51).

q

Calculated by triglycine – diglycine, from Ref(51).

r

Calculated by diglycine – glycine, from Ref(54).

s

Calculated by triglycine – diglycine, from Ref(54).

t

Calculated by tetraglycine – triglycine, from Ref(54).

u

Calculated by (triglycine – glycine)/2, from Ref(54).

v

Calculated by (tetraglycine – diglycine)/2, from Ref(54).

w

Calculated by (tetraglycine – glycine)/3, from Ref(54).

x

Calculated by diglycine – glycine, from Ref(59).

y

Calculated by triglycine – diglycine, from Ref(59).

z

Calculated by leucine – glycine, from Ref(59).

10

Tanford‘s Transfer Model, depicted in Scheme 1, is a thermodynamic cycle in which the horizontal reactions represent the native (N) to unfolded (D) equilibria of a protein with free energy changes of GNWD and GNOSD in water and in an osmolyte solution, respectively (60). The vertical reactions illustrate the transfer of the native and unfolded proteins from water to the osmolyte solution, with the respective transfer free energies denoted as ΔGtr, N and ΔGtr, D.

Scheme 1. Tanford‘s Transfer Model Rearranging, GNOSD  GNWD  Gtr ,D  Gtr , N , one obtains, GNOSD  GNWD  Gtr , D  Gtr , N . The differential transfer free energy of the protein in the

native and the unfolded states, Gtr , D  Gtr , N , can be equated to the m[osmolyte] term of the linear extrapolation method (LEM) (52):

GNOSD  GNWD  m[osmolyte]

[8]

At [osmolyte] = 1 M, the slope of the dependence of GNOSD on [osmolyte], m, is identical to the difference in the transfer free energies of the protein in the native and unfolded states, i.e. m 

Gtr ,D  Gtr , N 1M

.

Bolen and coworkers used their values of Δµtr‘ determined for amino acid side chains and the peptide backbone to predict the m-value for proteins in binary solvents containing 1 M of osmolyte using:

11

mcalc  Gtr , D  Gtr , N 

 n 

i i  AAType

sc tr ,i

 isc   trbb

 n 

i i  AAType

bb i

[9]

sc where ni is number of residues of type i in the protein;  tr, and  trbb are free energies of i

the transfer of the amino acid side-chains and peptide group from water to 1M osmolytes;  isc and  ibb are the fractional changes in solvent accessibility of amino acid

side-chains and peptide group associated with protein denaturation calculated according to the algorithm presented in Ref(52). For a set of globular proteins, Bolen and coworkers compared calculated and experimental m-values for various stabilizing and destabilizing osmolytes (52,61). A good agreement between the predicted and the experimentally-determined m-values was found. In these studies, the sum of the peptide contributions to GNOSD was determined to be more significant than the side chain contributions. Hence, it was concluded that the peptide units predominately control the folding/unfolding transitions of proteins (52,53,61,62). The free energy of transferring the peptide bond from water to a binary solvent containing a stabilizing osmolyte, e.g. glycine betaine, is positive, while the transfer to a solution of destabilizing osmolyte, e.g. urea, is accompanied by a negative change in free energy (52,53,61,62). Bolen and coauthors reported that the collective side-chain contribution to transfer free energy is either zero or opposite to that of the peptide bond, depending on the osmolyte type (52,53,61,62). In urea, the collective side-chain Gtr contribution is positive, where the contribution of the peptide bond is negative (61). The majority of the unfavourable interaction is attributed to ionizable side-chains (61). Polar side-chains are indifferent, while non-polar side-chains interact favourably with urea (61). In solutions of glycine betaine, peptide bonds interact unfavourably with the cosolvent, dominating over the collectively favourable interactions of the side-chains (61). Although solubility studies in combination with the Tanford transfer model have gained considerable recognition in the field of protein folding, the methodology contains several points that may compromise the validity of the conclusions reached. The experiments are performed at the solubility limit of the solute (up to several molar). At 12

such elevated concentrations, not only will solute-solvent interactions be present, but also solute-solute interactions. Protein studies, however, are performed at much lower concentrations (~1 mM or less). In addition, while some amino acids are very well soluble (e.g. glycine or serine), others are only sparingly soluble (e.g. tyrosine). Consequently, the extent of solute-cosolvent interactions at the solubility limit of the solute (amino acid) in 1 M cosolvent may vary significantly from one amino acid to another. Understandably, such data, if applied to proteins, may produce unaccountable errors. Except for glycine and alanine, the activity coefficients of all other amino acids are unknown. Therefore, only the apparent free energy of transfer, Δµtr‘, can be determined (61). Bolen‘s laboratory calculated both the true free energy of transfer, Δµtr, and the apparent free energy of transfer, Δµtr‘, for glycine and alanine in urea and they found that Δµtr, and Δµtr‘, differ significantly (53). These authors observed a much better agreement with experimental m-values calculated using the activity coefficient-corrected values of Δµtr for glycine (53). Although the corrected values of Δµtr have been used for glycine and alanine in the additive scheme, the apparent transfer free energies, Δµtr‘, for other amino acids contain unaccounted thermodynamic non-ideality effects. Therefore, the use of sc these Δµtr‘ in determining the side-chain free energy contributions,  tr, , may produce i

significant errors (7,8). Furthermore, volumetric studies have examined the use of various model compounds to extract the partial molar volume and adiabatic compressibility contributions of the peptide backbone and amino acid side chains (7,19,63). It was found that zwitterionic amino acids make poor models for mimicking the solvation properties of amino acids in proteins because the solvation shells of the side-chains interact with the solvation shells of the charged termini (19). Notably, the importance of the choice of low molecular weight model compound has been observed for the apparent transfer free energy, Δµtr‘ (58). The values of Δµtr‘ determined using different model compounds may be significantly different (see Table 1-1). The difference in Δµtr‘ may originate from the interactions via the overlap of the solvation shells of the side-chains and other functional groups of the molecule and from unaccounted thermodynamic non-ideality effects due to the lack of data on activity coefficient. Another limitation to solubility experiments is that it cannot be applied directly to proteins as the high protein concentration requirement could induce aggregation. As such, the group-wise contributions calculated using Δµtr for amino

13

acid side chains and peptide backbone can be compared only against the m-values and not directly with protein solubility data. Since the linear extrapolation method (LEM) is applicable to only certain osmolytes, solubility studies can be validated only with osmolytes in which the linear extrapolation method (LEM) is applicable (5,64).

1.4 Vapor Pressure Osmometry and Local-Bulk Solute Partitioning Model Provide Valuable Insights into Osmolyte-Protein Interactions One of the most extensive set of studies probing the interactions between osmolytes and proteins comes from Record‘s laboratory. In 1996, Record and coworkers developed novel approach of vapor pressure osmometry to quantify the interactions between osmolytes and proteins (65). Vapor pressure osmometry measures the osmolality of a solution, Osm, which can be interpreted as a nonideality-corrected effective total concentration of solute that directly relates to the thermodynamic activity of water, i.e. Osm = -55.5ln a1, (9). In this approach, the solution osmolality is determined as a function of osmolyte molality in the presence and absence of protein in order to determine the preferential interaction parameter, Г23, equivalent to that measured from equilibrium dialysis experiments (66). Specifically, the excess osmolality, ΔOsm, of the solutions containing proteins are determined to obtain the derivative of the chemical potential of the protein with respect to the molality of osmolyte, µ23, via the following relationship (9):

23  RT

Osm m2 m3

[10]

where m2 is the molality of the protein; m3 is the molality of the osmolytes; T is temperature; R is the universal gas constant; Osm(m2, m3) is the osmolality of an aqueous solution containing the protein and osmolyte at molal concentrations of m2 and m3, respectively. The excess osmolality of the solution, defined as ΔOsm = Osm(m2, m3) - Osm(m2, 0) - Osm(0, m3), is a measure of the favourable or unfavourable interactions of two nonelectrolyte solute components relative to their interactions with water (9). Osm(m2, 0) represents the osmolality of an aqueous solution containing the protein at a molal 14

concentration of m2 in the absence of osmolytes; and Osm(0, m3) is the osmolality of a binary solvent containing an osmolyte at a molal concentration of m3 (67). Record and coworkers quantified the extent of exclusion or accumulation of urea and glycine betaine near the protein surface by determining the preferential binding parameter of proteins, calculated via Г23 = -µ23/ µ33, where µ33 ≡ (∂µ3/∂m3)T,P,m2(68). In one of the earlier studies, Record and coworkers quantified the interactions of urea with native bovine serum albumin surface as a function of nondenaturing concentration of urea (69). They found that the Г23 is positive and depends linearly on the molal concentration of urea in the bulk, m3bulk , over the range of 0 to 1 mol/kg, with a slope, 23 / m3bulk , of 6 ± 2 (69). Thus, urea is accumulated in the vicinity of bovine serum albumin, an observation in agreement with the results from Timasheff‘s laboratory (44). In a subsequent study, using hen egg white lysozyme and bovine serum albumin as model proteins to study the interactions between glycine betaine and folded proteins, Record and coworkers found that Г23 is a linear function of m3bulk with the slopes, 23 / m3bulk , for bovine serum albumin and lysozyme being equal to -23.1±1.4 and -3.1±1.1, respectively (67). Hence, glycine betaine is excluded from each of the two protein surfaces (67). These values are consistent with the values of dГ23/dm3 of -26.1 ± 7.0 for bovine serum albumin and -4.0 ± 1.4 for lysozyme, that can be estimated from the data of Arakawa and Timasheff (47). For comparing different proteins varying in size, Record and coworkers normalized 23 / m3bulk per solvent accessible surface area, ASA, of the protein (67). The values of

Г23/( m3bulk ASA ) for relatively uncharged of lysozyme and bovine serum albumin, highly charged lacI helix-turn-helix DNA binding domain, and polyanionic calf thymus DNA are (-0.47 ± 0.17) x 10-3 m-1 Å-2, (-0.83 ± 0.05) x 10-3 m-1 Å-2, (-0.38 ± 0.05) x 10-3 m-1 Å-2 and (-1.8 ± 0.1) x 10-3 m-1 Å-2, respectively (67). A comparative analysis of these data reveals that the extent of exclusion of glycine betaine per Å2 of biopolymer area increases with the proportion of charge present on the surface (67). The values of Г23/( m3bulk ASA ) for bovine serum albumin and lacI helix-turn-helix DNA binding domain have been interpreted in terms of using the local-bulk solute partitioning

15

model to quantify the extent of accumulation or exclusion of urea and glycine betaine (66,69,70). The local-bulk solute partitioning model separates the protein solution into the local domain and the bulk domain (71). The local domain is a thermodynamically defined region in the vicinity of the protein surface, while the bulk domain is a region sufficiently distant from the protein so that the distribution of osmolytes and water are not perturbed by the presence of the protein (71). The local-bulk solute partitioning model defines the local bulk partition coefficient as K p  m3 / m3 . It is related to Г23/( m3bulk ASA ) according to the

following relationship: ( K p  1)b1o 23  m3bulk ASA m1

[11]

where m3local and m3bulk are the molal concentrations of the osmolyte in the local and bulk domains; b1o is the average number of water molecules in the local domain per Å 2 of protein surface, which is set to 0.11 H2O/ Å2 (i.e. 9 Å2/ H2O); and m1 = 55.5 mol of water/kg of water (71). A Kp greater than unity indicates the accumulation of osmolytes at the protein surface, whereas a Kp between 0 and 1 is a signature for exclusion. For native bovine serum albumin in urea, the Kp is 1.10 ± 0.04, indicating that urea is mildly accumulated at the protein surface (69). In glycine betaine, the Kp for bovine serum albumin is 0.14, suggesting an exclusion of glycine betaine from the vicinity of the protein (66). The Kp for lacI helix-turn-helix DNA binding domain exhibits a weak dependence on urea concentration up to 6 M urea while a Kp of 1.12 ± 0.02 demonstrates that urea is weakly accumulated at the surface (70). In glycine betaine, the value of Kp for the same protein increases steeply with the glycine betaine concentration, from 0.83 at 0 M glycine betaine to 1.0 at 4 M glycine betaine. At glycine betaine concentrations above 4 M, Record and coworkers predict that Kp will exceed 1, signifying glycine betaine accumulation and, hence, protein destabilization (70). To quantify the interactions of urea and glycine betaine with various functional groups found in proteins, Record‘s laboratory determined the chemical potential derivative, µ23, for a set of low molecular weight model compounds with different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, 16

and amide or cationic N atoms (68,72). The values of µ23 are dissected into additive contributions of group-specific solute-cosolvent interactions based on the water-accessible surface area, (ASA)i, of each functional groups (9,68).

 23 RT

   i ( ASA) i   ion  ion

[12]

i

where αi represents the normalized contribution of surface of type i (also known as the intrinsic interaction potential per unit area of functional group i);  ion is the stoichiometric number of ions released per formula unit of model compound; βion denotes the contribution of the interaction of urea or glycine betaine with an inorganic ion. The intrinsic interaction potential normalized per unit area of functional group i, αi, can be interpreted using a modified arrangement of the local-bulk partitioning model such that the partition coefficient, Kp,i, can be estimated:

i  

( K p ,i  1)b1,i (1   )

[13]

m1

The molality of water, m1, is set to 55.5 mol/kg, the number of waters of hydration per unit area, b1,i, is set to 0.18 H2O/ Å2, and ε is an osmolytes-osmolyte self-nonideality correction term defined as ε = d ln γ3/ d ln m3 (9,68). A comparative analysis of group-specific local-bulk partition coefficients, Kp,i, for glycine betaine and urea reveals that the extent of glycine betaine accumulation in the vicinity of the functional groups ranks in the following order: aromatic C (Kp = 1.62 ± 0.11) > amide N (Kp = 1.54 ± 0.19) > cationic N (Kp = 1.32 ± 0.11) > hydroxyl O (Kp = 0.97 ± 0.06) > aliphatic C (Kp = 0.92 ± 0.08) > amide O (Kp = 0.24 ± 0.27) > carboxylate O (Kp = 0.22 ± 0.06) > phosphate O (Kp = 0) (9,68). Urea accumulates in the vicinity of atomic groups in the following order: amide O (Kp = 1.28 ± 0.06) ~ aromatic C (Kp = 1.28 ± 0.02) > carboxylate O (Kp = 1.13 ± 0.05) > amide N (Kp = 1.10 ± 0.07) > hydroxyl O (Kp = 1.08 ± 0.02) > aliphatic C (Kp = 1.03 ± 0.02) > cationic N (Kp = 0.94 ± 0.05) (68). Record and coworkers have proposed that the key differences in the Kp values for interactions of urea and glycine betaine with N and O groups are due to urea‘s ability to donate and accept

17

multiple hydrogen bonds and glycine betaine‘s inability to serve as a hydrogen bond donor (68). The strong accumulation of urea in the vicinity of amide O and carboxylate O suggests that the hydrogen bond formed between the amide H of urea and the amide/carboxylate O group is more favourable than the hydrogen bond formed between the two O groups and water. Since glycine betaine lacks the capacity to serve as a hydrogen bond donor, water molecules preferentially interact with amide O and carboxylate O, while glycine betaine remains excluded from these groups. The accumulation of glycine betaine in the vicinity of amide N and cationic N indicates that glycine betaine is a better hydrogen bond acceptor from these groups compared to water. In contrary, urea molecules are only weakly accumulated around amide N compared to amide O, which indicates that the urea-NH…O=C-amide hydrogen bond is more favourable than the urea-NH…N-amide hydrogen bond. The mild exclusion of urea from cationic N indicates that the unfavourable urea-NH…N+(cationic N) interaction dominates over the more favourable (cationic N)N+…O=C-urea interaction. Aromatic carbons interact favourably with both urea and glycine betaine, as shown by accumulation of these osmolytes in the vicinity of aromatic C. In glycine betaine, this observation is explained by the cationic-π interaction of the trimethyl ammonium group of glycine betaine with the aromatic ring. In urea, the favourable interaction is ascribed to hydrogen bonding or partial cation- π interaction of the N groups on urea with the aromatic ring, and/or π- π stacking interactions between the π-system or urea and the ring (68,72,73). While vapour pressure osmometry measurements have provided valuable insights into the quality of the interactions urea and glycine betaine form with proteins and their functional groups, only three proteins have been studied using this approach (65–67,70). The small number of proteins studied imposes a barrier to generalize the findings to other proteins. Furthermore, the contributions from the interactions of urea or glycine betaine with the individual functional groups of the model compound are found by dissecting the model compounds based on the water-accessible surface area of the functional group (9,68,74). Dill warns that one should be cautious in employing group additivity in quantifying non-covalent interactions because each group may not necessarily reside in a uniform neighbouring environment (75). For example, the hydrophobicity of hydrophobic

18

patches on a protein surface depends on the chemical and topographical context of their surroundings (76). Using molecular dynamic simulations on self-assembled monolayers exhibiting either –CH3 groups or –OH groups on the surface, Garde‘s group showed that introducing a single –OH head in a –CH3 background has a greater effect on the density fluctuation of water at the self-assembled monolayer-water interface than adding a single –CH3 group in a –OH background (77). As such, one should take into consideration of the local effects around a functional group in a context-dependent manner while assimilating the group contributions to interpret osmolytes-protein interactions. In addition, vapour pressure osmometric measurements rely significantly on the local-bulk domain model for interpretation (73). This model, similar to Timasheff‘s interpretation of the preferential interaction parameter, defines the vicinity of the solute as a domain in which the solute exercises a thermodynamic influence on osmolyte and water molecules (73). The local-bulk domain model is limited in that it does not specify the geometric region to which osmolytes are interacting with the protein.

1.5 The Combination of Volumetric Measurements and the Solvent Exchange Model Is a Promising New Method for Studying Protein Solvation Volumetric observables such as the partial molar volume, compressibility, and expansibility of a solute directly reflect the entire spectrum of solute-solvent interactions present in the solution (18). These volumetric observables are, arguably, the most sensitive set of thermodynamic parameters for characterizing solute solvation (17). Importantly, these volumetric observables contain qualitative and quantitative information on the interactions of a solute with the principal solvent and cosolvent. However, the challenge is to extract this information and interpret it in terms of group-specific contributions of solvation to the stabilization/destabilization for proteins (17). To this end, the Chalikian laboratory has recently developed a novel method of probing solute–cosolvent interactions based on a combination of high precision volumetric measurements and a statistical thermodynamics formalism (7,78). The volumetric measurements are analyzed within the framework of a statistical thermodynamic model in

19

which the association of a cosolvent with a hydrated solute is accompanied by a release of r water molecules from the hydration shell of the solute to the bulk (7,78): SW r + C  SC + rW

Scheme 2

where S, C, and W signify the binding site, cosolvent, and water, respectively. If a solute has n water-binding sites, n/r is the maximum number of cosolvent binding sites. Under the assumption of n/r identical and independent cosolvent binding sites, a change in volume associated with the transfer of a solute from water to a concentrated cosolvent solution, ΔVo, is given the relationship (7,78): V ( nr )( a 3r )k a

V  VC   1nV o

o 1



1

1  ( a 3r )k a

[14]

1

where a1 and a3 are the mole fraction activity of water and molar activity of the cosolvent, respectively; ΔVC is the differential volume of the cavity enclosing a solute in a concentrated cosolvent solution and water (VC in a cosolvent solution minus VC in water); k is the effective equilibrium constant for the reaction in which a cosolvent molecules replaces r water molecules by binding to each of the n/r binding sites (see scheme 2); ΔV = ΔV0 + γ1rΔV°1 −γ3ΔV°3 is the change in volume associated with the cosolvent-induced replacement of water of hydration in a concentrated cosolvent solution; ΔV0 is the solvent exchange volume in an ideal solution; ΔV°1 and ΔV°3 are the excess partial molar volumes of water and cosolvent in a concentrated solution, respectively; and γ1 and γ3 are the correction factors reflecting the influence of the bulk solvent on the properties of the solvating water and cosolvent, respectively (7,78). Similarly, a change in isothermal compressibility accompanying the water-to-cosolvent transfer of a solute, ΔK°T, is described by the relationship: KT ( nr )( a 3r )k a

K

o

T

 KTC   1nK

o

T1



1

1  ( a 3r )k a

1

V 2 ( nr )( a 3r )k a



1

RT [1  ( a 3r )k ]2 a

1

20

[15]

where ΔKTC = −(∂ΔVC/∂P)T is the differential compressibility of the cavity enclosing a solute in water and a concentrated cosolvent solution; ΔKT = ΔKT0 + γ1rΔK°T1 − γ3ΔK°T3 is the change in compressibility associated with the binding-induced replacement of water of hydration with cosolvent; ΔK°T1 and ΔK°T3 are the excess partial molar isothermal compressibilities of water and cosolvent in a concentrated cosolvent solution, respectively; and ΔKT0 = −(∂ΔV0/∂P)T is the change in compressibility associated with solvent replacement in an ideal solution. Given the large heat capacity and small expansibility of water-based solutions, the difference between the partial molar adiabatic and isothermal compressibilities of a solute is small and, therefore, can be ignored. Consequently, partial molar adiabatic compressibilities instead of the partial molar isothermal compressibilities can be used in the formalism (7,78). The effective equilibrium constant, k, determined from the fitting of volumetric data is a fundamental thermodynamic parameter characterizing solute-cosolvent interactions. The effective equilibrium constant can be used to find the differential free energy of solute-solvent interactions, ΔΔGI, via the following relationship (7,78):



GI  ( nr ) RT ln ( aa1 ) r  k ( aa3r ) 10

10



[16]

where a10 is the mole fraction activity of pure water which is unity by definition. A change in free energy accompanying the transfer of a solute from water to the binary solvent, ΔGtr, is given by the sum of differential interaction free energy, ΔΔGI, and the differential free energy of cavity formation, ΔΔGC:



Gtr  GC  GI  GC  ( nr ) RT ln ( aa1 ) r  k ( aa3r ) 10

10



[17]

The differential free energy of cavity formation, ΔΔGC, can be estimated using scaled particle theory (SPT) (7). The advantages of studying solute-solvent interactions in a binary solvent using a combination of volumetric measurements and the solvent exchange model include:

21

(1) No restriction on the size of solute. The method works well for both proteins and low molecular weight model compounds. (2) No limitations on solvent composition. The concentration of cosolvents can be low or high, provided that the solute can dissolve in the solvent. (3) Applicable to a wide range of pH. Volumetric measurements can be made over the entire range of biologically relevant pH. (4) The measurements can be made at temperatures within the range of ~10° to ~70°C. (5) The solute is present at a low concentration. This allows for the determination of the solute-solvent and solute-cosolvent interactions with minimal interference from solute-solute interactions. Lee and coworkers have recently employed a combination of volumetric measurements and the solvent exchange model to study the interactions between urea and protein functional groups (7). They measured the partial molar volume and adiabatic compressibility of N-acetyl amino acid amides, N-acetyl amino acid methylamides, N-acetyl amino acids, and oligoglycines in aqueous solutions of urea at concentrations ranging from 0 M to 8 M. These measurements were dissected into volumetric contributions of the amino side chains and the glycyl unit using the additivity principle. These data were subsequently analyzed within the framework of the solvent exchange model to determine the effective equilibrium (binding) constants, k, for the reaction in which urea binds to the glycyl unit and each of the naturally occurring amino acid side chains replacing two waters of hydration. The effective equilibrium constants, k, ranging from 0.04 to 0.39 M, indicate that urea binds directly to a wide range of protein groups. Furthermore, the effective equilibrium constants, k, were used to determine the differential free energy of solute-solvent interactions, ΔΔGI, in urea and water. The observed range of equilibrium constants, k, corresponds to the values of ΔΔGI ranging from highly favourable to slightly unfavourable. In aggregate, these data support a direct interaction model in which urea denatures a protein by concerted action via favourable interactions with a wide range of protein groups (7).

22

1.6 Proposed Mechanisms of Urea-Induce Protein Denaturation Two mechanisms have been proposed to explain how urea unfolds proteins (79,80). In the indirect mechanism, denaturants are presumed to disrupt the structure of water thereby weakening the hydrophobic effect (79,81,82). Many experimental and computation studies, however, found that urea can incorporate into the hydrogen-bonded network of water without perturbing the spatial distribution of water molecules around urea (83,84). Hence, the notion that urea denatures protein through perturbation of water is rather controversial (85). The second mechanism, known as the ―direct mechanism‖, implies that direct van der Waals or hydrogen bonding or other electrostatic interactions exist between urea and protein groups (86–88). Numerous experimental and molecular dynamics simulation studies support the direct mechanism (7,10,53,69,86,89). Even among the proponents of the direct mechanism, however, there is a lack of consensus on the precise mechanism of urea-induced protein denaturation. Molecular dynamics simulations conducted by Thirumalai‘s group indicate that protein unfolding in urea is driven by hydrogen-bonding interactions between urea and peptide carbonyl groups and charged side chains of proteins (84,86,90,91). Thirumalai‘s group found no evidence that hydrophobic interactions have any major contribution to urea-induced protein denaturation (83,90). Furthermore, Thirumalai and coworkers‘ molecular dynamics simulations suggest that urea does not disrupt the structure of water even at a high urea concentration (83,84). In contrast to Thirumalai‘s results, molecular dynamics simulations performed by Grubmuller‘s group show that hydrophobic interactions, rather than hydrogen-bonding interactions, are the main driving force in urea-induced protein denaturation (89,92). A molecular dynamics simulations study on tripeptides in urea revealed that urea mainly interacts with nonpolar and aromatic side chains and peptide group while the polar and charged residues interact less frequently with urea (89). For amino acid side chains that are capable of forming hydrogen-bonding interactions, Grubmuller‘s group found that the hydrogen bonds formed between these amino acid side chains and water are more favourable than with urea (89). Grubmuller and coworkers proposed that the

23

accumulation of urea in the vicinity of the less polar amino acid side chains and exposed backbone accompanies a concomitant displacement of water molecules from the solvation shells, which is both entropically and enthalpically favourable (89). This, in effect, weakens the hydrophobic effect, making the unfolding of the protein more favourable. Hydrogen bonds formed between urea and the peptide backbone are not the main driving force for protein unfolding (89,92). PNIPAM is a model compound mimicking generic proteins. However, it contains only the peptide moiety and is devoid of charged and polar residues. Measurements of PNIPAM in urea, monomethylurea, dimethylurea, and trimethylurea showed that the direct binding of urea led to cross-linking and the stabilization of the collapsed state (81). In agreement with Grubmuller‘s results (89,92), Sagle and coworkers conclude that urea does not unfold proteins via direct hydrogen-bonding interactions with the proteins (81).

1.7 Proposed Mechanisms of Glycine Betaine-Induce Protein Stabilization The osmoprotective characteristics of glycine betaine have been recognized for over three decades and numerous attempts have been made by experimental and theoretical research laboratories to characterize the mode of action of glycine betaine on the stability of proteins (2,9,47,61,93,94). Nevertheless, there is no consensus to the precise mechanism of action of glycine betaine in protein stabilization. Similar to urea studies, two mechanisms have been proposed to explain how glycine betaine stabilizes the native conformation of proteins; namely, the direct mechanism and indirect mechanisms (9,47,61,68,93,94). An early ―direct hypothesis‖ to explain glycine betaine‘s ability to stabilize proteins has been proposed by Timasheff (47). Since the surface of the native globular protein is highly polar, Timasheff has deduced that the hydrophobic methyl groups of glycine betaine are not likely to interact with the protein surface (47). The charged groups of glycine betaine favour forming interactions with water over forming interactions with polar groups on the protein surface. Consequently, the protein preferentially interacts with

24

water which leads to the observed preferential hydration of proteins in glycine betaine solutions (47). More recently, based on amino acid solubility measurements, Bolen‘s group proposed that the main driving force that stabilizes the native state of globular proteins in glycine betaine solutions results from the unfavourable interaction between glycine betaine and the peptide backbone (61). Glycine betaine interacts favourably with most amino acid side chains (61). In the native state, these interactions slightly dominate over the unfavourable interactions between glycine betaine and the peptide groups, lowering the free energy of the native conformation of the protein (61). In the unfolded state, however, exposure of peptide groups to glycine betaine causes a net increase in free energy, rendering the unfolded conformation energetically unfavourable in the glycine betaine. The net increase in the free energy of the unfolded state in the presence of glycine betaine shifts the equilibrium toward the folded state (61). Record and coworkers have rationalized their vapour pressure osmometric measurements on model compounds using a combination of the additivity principle and the local-bulk solute partition model (9,68,74). The extent of glycine betaine accumulation in the vicinity of atomic groups decreases in the following order: aromatic C > amide N > cationic N > hydroxyl O > aliphatic C > amide O > carboxylate O > phosphate O (9,68). The interaction between glycine betaine and hydroxyl O is unfavourable; glycine betaine is excluded from the surface of hydroxyl O and from all the atomic groups beyond hydroxyl O in the ranking (9,68). Record and coworkers have ascribed the unfavourable interactions between glycine betaine and the oxygen atoms from amide, carboxylate and phosphate groups to glycine betaine‘s inability to compete with water to form hydrogen-bonding interactions with these oxygen containing groups (9,68). The favourable interactions between aromatic carbons and glycine betaine are due to cationic nitrogen-π interactions (9,68). The cationic nitrogen from quaternary ammonium or guanidinium group forms either a hydrogen bonded ion pair or salt bridge with the anionic carboxylate of glycine betaine (9,68). The favourable interaction of glycine betaine with amide nitrogen likely indicates that the

25

amide hydrogen is a better hydrogen bond donor than water; hence, the anionic carboxylate of glycine betaine preferentially accepts the hydrogen bond donated by the amide (9,68). Taken together, the works of Timasheff, Bolen and Record suggest a direct mechanism of the stabilizing action glycine betaine (9,47,61,68). Glycine betaine has been reported to perturb the hydrogen bonding network of water (93,94). Molecular dynamics simulations of the radial distribution function of water oxygen-water oxygen, hydrogen bond length distribution, hydrogen bond angle distribution, and hydrogen bond autocorrelation function between water molecules in the presence and absence of glycine betaine reveal that the hydrogen bonding network of water strengthen in the presence of glycine betaine (93). Results of these simulations suggest that the strengthening of the hydrogen bonding network of water due to the presence of glycine betaine increases the stability of the native conformation of proteins (93). Saladino and coworkers studied the human villin headpiece C-terminal helical subdomain (HP35) protein in the presence of glycine betaine using a combination of molecular dynamics simulations, circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, and developed an alternative ―indirect hypothesis‖ to explain the mechanism of the stabilizing action of glycine betaine (94). These authors calculated the solvent density function (SDF) to estimate the spatial arrangement of glycine betaine around the HP35 protein (94). The calculated SDF shows no relevant peak to support preferential interactions of glycine betaine (94). Consequently, it was concluded that glycine betaine makes no direct contact with the protein (94). The rotational correlation function suggests an increase in correlation times for water molecules in 1 M glycine betaine (94). Therefore, it has been proposed that the main driving force behind glycine betaine‘s stabilizing effect on the native conformation of proteins is a slowdown in the rotational diffusion of water in the presence of glycine betaine (94). The slowdown in solvent dynamics is accompanied by an apparent reduction in solvent polarity and dielectric constant, which resembles solvent at a cold temperature (94). This feature reduces the solvent‘s ability to participate in the interactions involved in the disruption of the native conformation. The slowdown in the rotational diffusion of water supports the hypothesis that glycine betaine exerts a

26

―water-structuring‖ effect (94).

1.8 Nucleic Acids Also Fold Into Globular Structures: DNA G-Quadruplex Guanine residues are capable of self-assembling into planar (or nearly planar) molecular squares known as G-quartets or G-tetrads (95). In a G-quartet, four guanine bases interact with one another through Hoogsteen base pairing, in which the N-7 and the oxygen at C-6 of the guanine serve as the hydrogen bond acceptors, while the N-1 hydrogen and the amino group at C-2 act as hydrogen bond donors (Figure 2).

Figure 1-2. Hoogsteen interaction in a G-quartet. The G-quartets are further stabilized by cations of the correct size that form coordination bonds with the lone pairs of electrons of the C-6 oxygen atoms. Stacked G-quartets fold into a complex structure known as G-quadruplex (96). A G-quadruplex is a four-stranded globular structure stabilized by stacks of two or more G-quartets with cations residing within the central cavity (22). Depending on its size, the cation may reside within the plane of the G-quartet (Na+), or between the G-quartets (K+). The extent of π-π stacking interactions between the quartets determines the degree to which the guanine residues shift from the usual planar conformation of G-quartets. (96). G-quadruplexes have sparked significant interest because many biologically important sequences contain consecutive guanine residues, which have been shown to fold in vitro

27

into G-quadruplex structures under physiological conditions (97). For example, G-rich repetitive sequences can be found in telomeres (98). Human telomeres which exhibit tandem repeats of the 5‘-(GGGTTA) motif spanning about 150 to 250 nucleotides shorten after every cell replication as a consequence of the end-replication effect (99). Telomeres, ultimately, reach a critical length and the cell undergoes apoptosis. In 80 to 85% of cancer cells and primary tumours, however, telomerase is overexpressed making these cells ―immortal‖ (99). Telomerase is a reverse transcriptase capable of adding nucleotides in the 3‘ direction lengthening the telomere beyond the critical length. One way to inhibit telomerase activity is to induce formation of G-quadruplex structures which would render the binding regions of telomeres inaccessible to telomerase (99). Recent bioinformatics studies have revealed that many G-rich DNA sequences in the promoter regions of genes have the potential to fold into G-quadruplex (100). Much effort is now devoted to studying the structural arrangements of G-quadruplex DNA and the factors stabilizing these complex structures (95).

DNA G-quadruplex can be unimolecular, bimolecular or tetramolecular and can adopt a variety of topologies (101). The topology and structure of a G-quadruplex depend on numerous factors, including the length and sequence of the motif, the size and nucleotide composition of the loops, the nature of the centrally bound cations, strand stoichiometry and alignment, hydration effects, and DNA concentration (29,102–104). Although there are no general rules to predict the topology of G-quadruplex and its stability based on the nucleic acid sequence, some common features have been observed. The stability of G-quadruplex structures increases with the number of successive G-quartets (102). Tran and coworkers studied a set of model oligonucleotides containing two, three or four consecutive guanines using ultraviolet (UV) melting, circular dichroism (CD) spectroscopic and non-denaturing PAGE measurements (102). With few exceptions, oligomeric sequences containing only two consecutive guanines do not form stable G-quadruplex structures over the temperature range from 10°C to 90°C (102). Of the d[(GGTTA)3GG], d[(GGCTTA)3GG], d[(GGTTAC)3GG], d[(GGTTACA)3GG], d[(GGTGTACT)3GG] sequences, each containing four repetitive telomeric motifs with two consecutive guanine residues, only d[(GGTTA)3GG] and d[(GGCTTA)3GG] form a small

28

fraction of intramolecular G-quadruplex structures in the presence of Na+ and K+ ions (102). Furthermore, Tran and coworkers‘ measurements are in agreement with the picture in which the sequence of the oligonucleotide is the determining factor in the formation of G-quadruplex structures. For example, d[(GGCTTA)3GG] and d[(GGTTAC)3GG] differ only in the position of the cytosine nucleotide relative to TTA, yet the former oligonucleotide folds into a G-quadruplex structure while the latter does not (102).

1.9 DNA G-quadruplex-Cation Interaction Is Important for Stabilization A general requirement to forming a stable G-quadruplex is the presence of cations capable of forming coordinate bonds with the C-6 carbonyl oxygen of the guanine residues in the central cavity. In a solid-state multinuclear NMR study, Wong and Wu determined the relative affinity of monovalent cations for a G-quartet structure formed by guanosine 5‗-monophosphates (5‗-GMP) (105). The binding affinity of monovalent cations is ranked in the order K+ > NH4+ > Rb+ > Na+ > Cs+ > Li+ (105). For divalent cations, the following order of stability, Sr2+ >>Ba2+ > Pb2+ > Ca2+ > Mg2+ > Co2+ > Zn2+, has been proposed (106). Cations dissolved in aqueous solutions are fully hydrated and, thus, they must first shed their hydration shells to enter the central cavity and form coordinate complexes with guanine residues. An ion with a lower charge density is dehydrated more easily and hence, G-quadruplexes preferentially bind K+ over Na+ (101). This is consistent with experimental data which suggest that G-quadruplexes containing three or four successive G-quartets exhibit higher values of TM in the presence of K+ rather than Na+ ions (102). Recently, Kim and coworkers assessed the contributions of the specific (coordination in the central cavity) and nonspecific (condensation) ion binding in G-quadruplexes by studying the change in TM of preformed G-quadruplexes following the addition of nonstabilizing ions Li+, Cs+, and TMA+ (tetramethylammonium). Their data suggest that the predominant ionic contribution to G-quadruplex stability comes from the specifically bound Na+ or K+ ions and not from counterion condensation (107). The G-quadruplex structures formed are highly dependent on the type of cations present. For a given sequence, the topology may differ depending on the identity of the 29

central

cation

(102).

For

example,

the

human

telomeric

oligonucleotide,

d[A(GGGTTA)3GGG], is predominantly in a basket structure featuring antiparallel strands with one diagonal and two lateral loops in a Na+ solution whereas, in the presence of K+, the same oligonucleotide exists as a mixture of at least two conformers. One of these conformers, characterized by X-ray crystallography, is a parallel-stranded propeller structure with three double-chain reversal loops (108). The second conformer, derived from solution NMR measurements, features a hybrid conformation comprised of three parallel strands and one antiparallel strand linked by loops on edges (109).

1.10 Hydration is a Major Determinant of DNA G-Quadruplex Stability Hydration is an important determining factor that regulates the thermodynamic stability of all DNA structures including G-quadruplexes (17,21). Circular dichroism (CD) spectroscopic measurements of DNA G-quadruplexes in the presence of cosolvents reveal that the thermal stability of the DNA G-quadruplexes is dependent on the concentration of the cosolvent (21). In particular, the G-quadruplex formed by thrombin-binding aptamer (TBA), d[G2T2G2TGTG2T2G2], exhibits an increase in thermal stability as the concentration of poly(ethylene glycol) (PEG 200) increases (21). An increase in thermal stability of the TBA G-quadruplex has been also detected in the presence of 1,2-dimethoxyethane, 2-methoxyethanol, 1,3-propanediol, and glycerol (21). In general, the conformation of DNA G-quadruplex depends on the type of cosolvent present (29). Using a combination of CD and NMR measurements, Miller and coauthors have reported that human telomeric G-quadruplex, d[AG3(TTAG3)3], exhibits different conformations in PEG 400 and in 50% (v/v) acetonitrile (29). To discriminate between the effects of hydration and molecular crowding on G-quadruplex stability, Miller and coworkers added 300 mg/mL of bovine serum albumin (BSA) to the human telomeric G-quadruplex and monitored the DNA conformation using NMR spectroscopy (29). This high BSA concentration amounts to only about 4.5 mM and therefore this solvent composition served to create molecular crowding while minimally affecting the water activity (29). At neutral pH, BSA is negatively charged and is thus unlikely to exhibit extensive binding to the negatively charged G-quadruplex. The NMR 30

spectrum of the G-quadruplex in BSA exhibits more resemblance to the spectrum of the quadruplex in buffer alone than in 50% (v/v) acetonitrile (29). It has, therefore, been concluded that the G-quadruplex is stabilized by dehydration and not by molecular crowding (29). Despite the important role of hydration in regulating the conformational preferences of nucleic acids, a survey of the literature reveals that studies of hydration changes accompanying DNA G-quadruplex-to-single strand transitions are scarce (110). The majority of such studies employs the osmotic stress technique in which the activity of water, a1, is modulated by adding a preferentially excluded cosolvent, while the apparent equilibrium constant, K, of the reaction studied, i.e. heat-induced G-quadruplex-to-coil transition, is measured as a function of a1 (21,28,31,111). The slope, (∂ln K/∂ln a1), depends on the reaction-induced changes in the number of water, Δn1, and cosolvent, Δn3, molecules preferentially associated with/excluded from the reactants, according to the following relationship: (∂ln K/∂ln a1) = Δn1 − (N1/N3)Δn3

[18]

where N1 and N3 are the numbers of moles of water and cosolvent present in solution, respectively (112). The key ambiguity of interpretation of osmotic stress measurements results from the necessity to separate the hydration term, Δn1, and the cosolvent binding term, -(N1/N3)Δn3, of Eq. (18). It is important to recognize that Δn1 and Δn3 in Eq. (18) are not necessarily constant, but may vary with an increase in cosolvent activity, a3, and an accompanying decrease in water activity, a1. Furthermore, Δn1 and Δn3 are dependent of each other in that, an increase in Δn3 would trigger a decrease in Δn1, in a manner that depends on the steric number of water molecules replaced by a cosolvent molecule upon its association with the DNA. The common practice in the osmotic stress studies is to rationalize the cosolvent-dependent G-quadruplex-to-single strand DNA equilibrium data solely in terms of Δn1 (21,28,31,111). Ignoring the effect of Δn3 may lead to misinterpretation of the osmotic stress measurements (112,113). With the assumption that the effect of Δn3 is negligible, osmotic stress measurements of helix-to-single strand DNA transitions have been interpreted to suggest

31

that the double-stranded conformation is more extensively hydrated compared to the single stranded state (21,112). A recent study of helix-to-single strand DNA transition using volumetric measurements, however, reveals that the single stranded state of DNA is more hydrated than the double stranded conformation. This finding is more conclusive than the osmotic stress studies because the interpretation of volumetric measurements does not have to deal with the effect of Δn3 (113). The volumetric approach is a direct method of sampling DNA hydration and changes in hydration accompanying various processes involving DNA (17). Volumetric observables such as partial molar volume, adiabatic compressibility, and expansibility are highly nonselective and, therefore, can probe the entire population of water molecules solvating a solute, regardless of the water molecules‘ distance relative to the solute, degree of immobilization, and localization (17). In fact, volumetric measurements can be applied to studying hydration of nucleic acids in the presence or absence of cosolvent, in a crowded or dilute solvent. In addition, volumetric techniques permit data collection at constant temperature or in a temperature-scanning regime. The flexibility, accuracy, precision, and sensitivity of partial molar volume and adiabatic compressibility measurements make them the method of choice for characterizing DNA hydration. While a number of volumetric studies of nucleic acids have been reported, no extensive volumetric and thermodynamic characterization of the folding/unfolding transitions of DNA G-quadruplex has been published. For this reason, we have employed a combination of volumetric, spectroscopic, and calorimetric measurements to characterize the thermodynamic and hydration properties of the Na+-stabilized antiparallel structure of the human telomeric oligonucleotide, d[A(GGGTTA)3G3], and the K+-stabilized hybrid-1 G-quadruplex structure of d[A3G3(T2AG3)3A2]. These measurements are of fundamental importance for understanding the role of water in the stability of DNA G-quadruplex structures.

1.11: Partial Molar Volume The volume of a solution, V, is defined as the partial derivative of Gibbs free energy, G, with respect to pressure, P, at constant temperature, i.e.

32

 G  V    P T

[19]

The partial molar volume, V°, of a solute is defined as the partial derivative of the solution volume, V, with respect to the number of moles, Ni, of the i-th solute at a constant temperature, T, pressure, P, and number of moles of the j-th solute, Nj‘:

 V   V o    N  i T , P, N j

[20]

When the derivative in Eq. (20) is taken at a very low concentration of solute i, the partial molar volume is defined as the partial molar volume at infinite dilution:

 V   Vo  lim Ci 0 N   i T , P , N j

[21]

The partial molar volume at infinite dilution can be interpreted as the apparent volume occupied by 1 mole of solute in solution at a negligibly low solute concentration. The expression for the apparent molar volume, φV, of solute i is:

V 

M





    

[22]

 C

where ρ and ρ0 are the densities of the solution containing solute i and the neat solvent, respectively; C is the molar concentration of solute i; and M is the molecular weight of solute i. At infinite dilution of i, the apparent molar volume is, within experimental error, identical to the partial molar volume at infinite dilution. Using scaled particle theory (SPT), the partial molar volume of a solute can be dissected into the following components: the intrinsic volume of a solute, VM, the thermal volume, VT, the interaction volume, VI, and βT0RT (114,115): V° = VM + VT + VI + βT0RT

[23]

33

where βT0 is the coefficient of isothermal expansibility of the solvent; R is the universal gas constant; and T is the temperature. The ideal term, βT0RT, is small, with a magnitude of 1.1 cm3 mol-1 at 25°C. This term is largely independent of temperature. The interaction volume, VI, is the change in volume due to the interactions between the solute, cosolvent, and solvent. The intrinsic volume, VM, is the geometric volume of a solute molecule that is not accessible to any part of solvent molecules. The VM of a small, fully solvent-accessible molecule can be approximated by its van der Waals volume, VW. The van der Waals volume can be calculated using the additive scheme and group contributions given in the literature (116,117). The intrinsic volume, VM, of larger molecules such as proteins and nucleic acids can be calculated from structural data using geometric algorithms presented by Richards (118). The thermal volume, VT, is the "void" volume around the solute arising from the steric, vibrational, and structural effects. The steric component of VT is a reflection of the imperfect packing of solute and solvent molecules in the solution. The vibrational component reflects the thermally-induced mutual vibrational motions of solute and solvent molecules. Theoretically, the vibrational component of VT should subside to zero at a temperature of absolute zero. The structural component originates from the open (tetrahedral) structure of water and related packing effects around solute molecules (119).

1.12: Partial Molar Adiabatic Compressibility The isothermal compressibility of a system, KT, is defined as:

 V  KT   TV     P T

[24]

where T is the absolute temperature; V is the volume of the system; P is the pressure; βT is the coefficient of isothermal compressibility, defined as:

34

 T 

1  V    V  P T

[25]

In a similar manner, the adiabatic compressibility of a system, Ks, is defined as:

 V  K S   SV     P  S

[26]

where S denotes entropy, and βS is the coefficient of adiabatic compressibility, defined as:

1  V   S    V  P  S

[27]

The coefficient of isothermal compressibility and the coefficient of adiabatic compressibility are related through the following relationship:

 T  S

T 2  cP

[30]

where cp is the specific heat capacity at constant pressure, and ρ is density. The coefficient of thermal expansion, α, is defined as:



1  V    V  T  P

[31]

The partial molar compressibility, K°, of a solute is defined as the partial derivative of the compressibility of the solution, K, with respect to the number of moles, Ni, of the i-th solute at a constant temperature, T, pressure, P, and number of moles of the j-th solute, Nj, i.e.

 K KTo   T  N i

  T , P , Nj

[32]

35

 K K So   S  N i

  T , P , Nj

[33]

K°T and K°S can be linked using the following relationship: T o KT  K   o cPo 2

o

o S

 2E o C o P       c o Po   o

[34]

where cpo is the specific heat capacity at constant pressure of the solvent; ρo is density of the solvent; αo is the coefficient of thermal expansion of the solvent; E° is the partial molar expansibility of the solute; and C°p is the partial molar heat capacity of the solute (120). Given the large heat capacity and small expansibility of water-based solutions, the difference between the partial molar adiabatic and isothermal compressibilities of a solute is small and, in many cases, can be ignored as a first approximation. The most accurate approach to determining the partial molar adiabatic compressibility is based on the Newton-Laplace equation (120):

U2 

1

S 

[35]

Upon differentiating the Newton-Laplace equation with respect to the molar concentration of the solute, C, and solving for the limit of C approaching zero, the following relationship can be derived:  M  K So   SO  2V o  2[U ]   o  

[36]

where M is the molecular weight of the solute; βSO represents the coefficient of adiabatic compressibility of the solvent; V° denotes the partial molar volume of the solute; and [U] is the relative molar sound velocity increment of the solute. The relative molar sound velocity increment of the solute is defined as:

36

[U ] 

U Uo U oC

[37]

where U and Uo are the sound velocities of the solution and the solvent, respectively.

1.13: Outline of the Thesis The work performed in my doctoral studies is devoted to understanding and quantifying solute-solvent interactions in the folded and unfolded states of proteins and DNA G-quadruplexes using volumetric techniques. Both globular proteins and DNA G-quadruplexes exhibit tertiary conformations with a globular geometry. In addition, hydration is a major driving force that governs the conformational preferences of both proteins and nucleic acids. As such, we have chosen to study both globular proteins and DNA G-quadruplex in parallel. To this end, we have designed a series of experiments to probe glycine betaine-protein interactions, solvation changes in urea-induced native-to-unfolded protein transitionsm and hydration changes accompanying DNA G-quadruplex-to-coil transitions. In Chapter 2, we employ volumetric measurements to characterize interactions of glycine betaine with protein groups. Specifically, we report the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides and oligoglycines at glycine betaine (GB) concentrations ranging from 0 to 4 M. Using these measurements, we evaluate the volumetric contributions of amino acid side chains and the glycyl unit (-CH2CONH-) as a function of GB concentration. These GB dependences are analyzed within the framework of a statistical thermodynamic model to evaluate the equilibrium (binding) constant, k, for the reaction in which a GB molecule binds each of the functionalities under study replacing four water molecules. We use scaled particle theory (SPT) to estimate the change in free energy of cavity formation, ΔΔGC. We subsequently use these equilibrium constants, k, to calculate the differential free energy of solute-solvent interactions, ΔΔGI, in a concentrated GB solution and water. We determine the free energy of the transfer of functional groups from water to concentrated GB solutions, ΔGtr, as the sum of ΔΔGC and ΔΔGI.

37

In Chapter 3, we present the first application of volumetric measurements to characterization of the interactions between glycine betaine and globular proteins. Specifically, we report the partial molar volumes and adiabatic compressibilities of cytochrome c, ribonuclease A, lysozyme, and ovalbumin in aqueous solutions of the stabilizing osmolyte glycine betaine (GB) at concentrations between 0 and 4 M. These globular proteins do not undergo any conformational transitions in the presence of GB which allows us to study the interactions of GB with proteins in their native states within the entire range of experimentally accessible GB concentrations. We analyze our volumetric results within the framework of a statistical thermodynamic model in which each instance of GB interaction with a protein is treated as a binding reaction accompanied by the release of four water molecules. This analysis enables us to evaluate the binding constants, k, and changes in volume, ΔV0, and adiabatic compressibility, ΔKS0, accompanying each GB-protein association event in an ideal solution. We compare these parameters with the equivalent parameters determined for low-molecular weight analogues of proteins in Chapter 2 to see if there are cooperative effects involved in interactions of GB with the proteins studied. We also evaluate the differential free energy of solute-solvent interactions, ΔΔGI, in a concentrated GB solution and water for the four proteins studied here. In Chapter 4, we report the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A in aqueous solutions containing 0 to 8 M urea. This set of proteins is chosen to model the full range of protein conformation states. At pH 7, lysozyme remains folded and is resistant to urea-induced denaturation. Apocytochrome c is unfolded between 0 and 8 M urea. Ribonuclease A and α-chymotrypsinogen A unfold in a two-state manner between 0 and 8 M urea. We interpret these volumetric dependences within a statistical thermodynamic framework that allows for the determination the equilibrium (binding) constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. This analysis permits us to estimate the solvent-accessible surface areas of the native and unfolded conformational states. We also compare the values of k, ΔV0, and ΔKT0 with the similar

38

parameters determined on small molecules mimicking protein groups to probe for cooperative effects involved in urea-protein interactions. In Chapter 5, we study the volumetric properties of the folded and unfolded states of an oligodeoxyribonucleotide containing four repeats of the human telomeric sequence, d[A(GGGTTA)3GGG]. This oligodeoxyribonucleotide folds into an antiparallel G-quadruplex in the presence of Na+. We employ a combination of pressure-perturbation calorimetry (PPC), vibrating tube densimetry, ultrasonic velocimetry, and UV melting under high pressure to characterize the volumetric changes associated with the coil-to-G-quadruplex transition in the presence of Na+. We also determine the change in the intrinsic geometric parameters of the ODN accompanying quadruplex formation using molecular dynamics simulation. We rationalize our experimental and computational results to determine the hydration contribution to the coil-to-G-quadruplex transition. In Chapter 6, we employ a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the human telomeric sequence, d[A3G3(T2AG3)3A2]. This oligodeoxyribonucleotide adopts the hybrid-1 G-quadruplex conformation in the presence of K+ ions. We monitor the kinetics of coil-to-G-quadruplex formation by measuring the time dependence of the ellipticity at 268 nm and 242 nm at a high and low concentration of oligonucleotides to assess the molecularity of the G-quadruplex formation process in the presence of K+. Furthermore, we interpret the volumetric and thermodynamic measurements in terms of the changes in hydration accompanying the folding transitions of this G-quadruplex. We compare the thermodynamic and volumetric results of this K+-stabilized G-quadruplex with the results obtained for the Na+-stabilized G-quadruplex state of d[A(GGGTTA)3GGG] discussed in Chapter 5 to gain insights on the conformational preferences of G-rich DNA sequences in the presence of Na+ and K+ ions. Chapter 7 summarizes the importance and the implications of the research presented in my dissertation. I also propose future studies extending upon the fundamental research conducted in my doctoral studies. Appendix A presents another published work that is not included in the earlier chapters of this dissertation.

39

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CHAPTER 2: Volumetric Characterization of Interactions of Glycine Betaine with Protein Groups

“Reprinted from Journal of Physical Chemistry B, 115, Yuen Lai Shek & Tigran V. Chalikian,. Volumetric characterization of interactions of glycine betaine with protein groups, 11481-11489. Copyright (2011), with permission from American Chemical Society”. Author‘s Contribution: Yuen Lai Shek performed all experiments, analyzed the data, and participated in the preparation of the manuscript.

52

2.1 ABSTRACT We report the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides and oligoglycines at glycine betaine (GB) concentrations ranging from 0 to 4 M. We use these results to evaluate the volumetric contributions of amino acid side chains and the glycyl unit (-CH2CONH-) as a function of GB concentration. We analyze the resulting GB-dependences within the framework of a statistical thermodynamic model and evaluate the equilibrium constant for the reaction in which a GB molecule binds each of the functionalities under study replacing four water molecules. We calculate the free energy of the transfer of functional groups from water to concentrated GB solutions, Gtr, as the sum of a change in the free energy of cavity formation, GC, and the differential free energy of solute-solvent interactions, GI, in a concentrated GB solution and water. Our results suggest that the transfer free energy, Gtr, results from a fine balance between the large GC and GI contributions. The range of the magnitudes and the shape of the GB-dependence of Gtr depend on the identity of a specific solute group. The interplay between GC and GI results in pronounced maxima in the GB-dependences of Gtr for the Val, Leu, Ile, Trp, Tyr, and Gln side chains as well as the glycyl unit. This observation is in qualitative agreement with the experimental maxima in the TM-versus-GB concentration plots reported for ribonuclease A and lysozyme.

53

2.2 INTRODUCTION Water-miscible cosolvents may dramatically affect the stability and thermodynamic state of proteins and nucleic acids.1-7 Small organic cosolvents are frequently referred to as osmolytes since their presence at high concentrations inside the cell is used by many organisms as protection against the osmotic loss of cellular water as well as other deleterious conditions such as elevated temperatures and pressures and desiccation.1,8 Cosolvents, in general, and osmolytes, in particular, are classified as protecting (stabilizing), neutral, or denaturing (destabilizing) depending on their mode of action. The latter can be assessed based on the sign of a change in free energy, Gtr, accompanying the transfer of a protein from water to a water-cosolvent mixture. One traditional way of evaluating Gtr is based on measuring the differential solubility of a solute under study in a water-cosolvent mixture and water.9 Such studies are, generally, conducted not on proteins per se but on relatively simple model compounds (such as amino acids) which mimic solvent-accessible protein groups.9 A change in free energy accompanying the transfer of a solute from the principal solvent (water) to a solvent-cosolvent mixture is the sum of the differential free energy of cavity formation, GC, and the differential free energy of solute-solvent interactions, GI, in a cosolvent solution and water.4,10,11

Gtr = GC + GI

(1)

54

It is generally accepted that the cavity term, GC, in Eq. (1) contributes unfavorably to the water-to-cosolvent transfer free energy, Gtr, while the contribution of the interaction term, GI, is favourable.4,12 The interplay between the cavity and interaction contributions is poorly understood. Multifaceted approaches combining theoretical and experimental methods are needed to elucidate the balance of forces which renders a specific cosolvent denaturing, protecting, or neutral. We have recently developed a volumetric technique in which the affinity of a cosolvent for a specific solute is evaluated based on the differential partial molar volume and compressibility measurements in solutions of model compounds mimicking a protein in water and water-cosolvent binary mixtures.11,13 We have applied such measurements to characterizing the interactions between the denaturing osmolyte urea and the 20 naturally occurring amino acid residues.11 In the present work, we extend these studies to the protective osmolyte glycine betaine (GB). To avoid the influence of the solute-induced shift in the ionization-neutralization equilibrium of GB on the measured volumetric observables, we limit our investigation to N-acetyl amino acid amides and oligoglycines lacking a net charge.

2.3 MATERIALS AND METHODS Materials. GB, glycine, diglycine, triglycine, tetraglycine, N-acetyl glycine amide, and N-acetyl tyrosine amide were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, Ontario, Canada). N-acetyl alanine amide, N-acetyl valine amide, N-acetyl leucine amide, N-acetyl isoleucine amide, N-acetyl proline amide, N-acetyl phenylalanine amide, N-acetyl tryptophan amide, N-acetyl methionine amide, and N-acetyl glutamine amide 55

were purchased from Bachem Bioscience, Inc. (King of Prussia, PA, USA). All amino acid derivatives were in L-stereoisomeric form. All the reagents used in the studies reported here were of the highest purity commercially available and used without further purification.

Solution Preparation. We prepared aqueous solutions of GB with concentrations of 1, 2, 3, and 4 M by weighing 10 to 50 g of GB and adding pre-estimated amounts of water to achieve the desired molalities, m. The molar concentration, C, of a GB solution was determined from the molal value, m, using C = [1/(mW) + V/1000]-1, where W is the density of water and V is the apparent molar volume of GB. The concentrated GB solutions were used as solvents for respective oligoglycines and amino acid derivatives. The concentrations of the samples were determined by weighing 10 to 20 mg of a solute material with a precision of ±0.02 mg and dissolving the sample in a known amount of solvent (GB solution). All chemicals were dried under vacuum in the presence of phosphorus pentoxide for 72 hours prior to weighing.

Determination of Partial Molar Volumes and Adiabatic Compressibilities of Solutes. Solution densities were measured at 25 °C with a precision of ±1.510-4 % using a vibrating tube densimeter (DMA-5000, Anton Paar, Gratz, Austria). The apparent molar volumes, V, of the solutes were evaluated from the relationship:

V = M/ - ( - 0)/(0m)

(2)

56

where M is the molecular weight of the solute; m is the molal concentration of the solute;  and 0 are the densities of the solution and the solvent (water or a GB solution), respectively. Solution sound velocities, U, were measured at 25 °C at a frequency of 7.2 MHz using the resonator method and a previously described differential technique.14-17 The analysis of the frequency characteristics of the ultrasonic resonator cells required for sound velocity measurements was carried out by a Hewlett Packard model E5100A network/spectrum analyzer (Mississauga, ON, Canada). For the type of ultrasonic resonators used in this work, the precision of sound velocity measurements is on the order of ±110-4 %.15,18,19 The acoustic characteristics of a solute which can be derived directly from ultrasonic measurements is the relative molar sound velocity increment, [U] = (U - U0)/(U0C), where C is the molar concentration of a solute, and U and U0 are the sound velocities in the solution and the solvent, respectively. The values of [U] were used in conjunction with the V values derived from densimetric measurements to calculate the apparent molar adiabatic compressibility, KS:

KS = S0 (2V - 2[U] - M/0)

(3)

where S0 = 0-1U0-2 is the coefficient of adiabatic compressibility of the solvent. The values of 0, U0, and S0 were directly determined for each GB solution from our densimetric and acoustic measurements. For each evaluation of V or KS, three to five independent measurements were carried out within a concentration range of 2 - 3 mg/mL.

57

Our reported values of V or KS represent the averages of these measurements, while the errors were calculated as standard deviations.

Determination of Excess Partial Molar Volume and Adiabatic Compressibility of Water. The excess partial molar volume of water in a GB solution is given by V°1 = V1 V°1, where V°1 = 18.07 cm3 mol-1 is the partial molar volume of pure water (in the absence of GB), while V1 is the partial molar volume of water in solution at a given GB concentration. The excess partial molar adiabatic compressibility of water is given by K°S1 = KS1 - K°S1, where K°S1 = 8.0910-4 cm3 mol-1 bar-1 is the partial molar adiabatic compressibility of pure water, while KS1 is the partial molar adiabatic compressibility of water at a given GB concentration. To determine the values of V1 and KS1,~0.1 g of water was added to ~10 g of each GB solution (with concentrations of 1, 2, 3, and 4 M) with the resulting changes in density and sound velocity being measured. The values of V 1 and KS1 were computed from Eqs. (2) and (3).

2.4 RESULTS Table 2-1 lists the values of 0 (column 2) and S0 (column 3) at 0, 1, 2, 3, and 4 M GB. The excess partial molar volume, V°1, and adiabatic compressibility, K°S1, of water measured at different GB concentrations are shown in Table 2-1 (columns 4 and 5, respectively). Tables 2-2, 2-3, and 2-4 present, respectively, the relative molar sound velocity increments, [U], apparent molar volumes, V, and adiabatic compressibility, KS, of the solutes investigated in this study at 0, 1, 2, 3, and 4 M GB. To the best of our

58

knowledge, no data of this kind have been reported. Therefore, our results cannot be compared with the literature. The apparent molar volumes and adiabatic compressibilities of oligopeptides and N-acetyl amino acid amides in water do not strongly depend on concentration.20-23 By extension, we assume that the concentration dependences of the volumetric properties of these solutes are insignificant also in concentrated solutions of GB, especially, given the diminutive solute concentrations used in this work. Consequently, we do not discriminate between the apparent molar volumetric characteristics the solutes studied in this work and their partial molar characteristics obtained by extrapolation to infinite dilution.

Table 2-1. Density of Solvent, 0, Coefficient of Adiabatic Compressibility of Solvent, S0, Excess Partial Molar Volume of water, V°1, and Excess Partial Molar Adiabatic Compressibility of Water, K°S1, as a Function of GB Concentration -----------------------------------------------------------------------------------------------------------[GB] M

0

S0

V°1

g cm-3

10-6 bar-1

10-3 cm3 mol-1

K°T1 10-4 cm3 mol-1 bar-1

----------------------------------------------------------------------------------------------------------0

0.997047

44.773

0

0

----------------------------------------------------------------------------------------------------------1

1.016001

39.610

0.1±0.6

-0.04±0.01

----------------------------------------------------------------------------------------------------------2

1.035113

34.946

7.1±2.2

-0.16±0.01

-----------------------------------------------------------------------------------------------------------

59

3

1.054651

30.841

23.2±0.8

-0.37±0.01

----------------------------------------------------------------------------------------------------------4

1.074620

27.161

53.5±2.2

-0.69±0.01

-----------------------------------------------------------------------------------------------------------

2.5 DISCUSSION Volume and Compressibility Contributions of Amino Acid Side Chains and Glycyl Unit. Tables 2-5 and 2-6 list, respectively, the volume and adiabatic compressibility contributions for the 10 amino acid side chains studied here and the glycyl residue as a function of GB concentration. The volume or compressibility contribution of a specific amino acid side chain was computed as the difference in the partial molar volume, V°, or adiabatic compressibility, K°S, between the corresponding N-acetyl amino acid amide and N-acetyl glycine amide. The volumetric contributions of the glycyl residue (-CH2CONH-) were obtained as the difference between the values corresponding to triglycine and tetraglycine.

Table 2-2. Relative Molar Sound Velocity Increments, [U] (cm3 mol-1), of Solutes as a Function of GB Concentration ----------------------------------------------------------------------------------------------------------Compounds

0M

1M

2M

3M

4M

----------------------------------------------------------------------------------------------------------N-Ac-Gly-NH2

33.8±0.2

28.7±0.5

22.5±0.5

16.1±0.7

9.2±0.4

-----------------------------------------------------------------------------------------------------------

60

N-Ac-Ala-NH2

43.3±0.2

37.5±0.3

30.8±0.2

24.5±0.7

16.7±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Val-NH2

59.9±0.3

53.1±0.5

44.1±0.2

34.1±0.4

23.7±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Leu-NH2

70.6±0.3

61.1±0.6

50.9±0.3

38.9±0.4

25.3±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Ile-NH2

70.4±0.2

63.0±0.4

54.4±0.6

45.1±0.4

36.2±0.8

----------------------------------------------------------------------------------------------------------N-Ac-Pro-NH2

54.5±0.3

48.1±0.5

40.4±0.5

33.4±0.4

26.4±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Phe-NH2

67.4±0.3

56.6±0.3

44.5±0.3

35.2±0.8

27.2±0.8

----------------------------------------------------------------------------------------------------------N-Ac-Trp-NH2

67.0±0.1

56.1±0.6

43.0±0.1

31.0±0.6

15.5±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Met-NH2

60.6±0.2

51.7±0.1

40.8±0.4

30.1±0.6

18.0±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Tyr-NH2

54.9±0.3

46.2±0.1

34.0±0.2

21.1±0.3

7.2±0.5

----------------------------------------------------------------------------------------------------------N-Ac-Gln-NH2

50.2±0.1

43.4±0.3

34.9±0.3

25.2±0.4

15.0±0.4

----------------------------------------------------------------------------------------------------------Glycine

35.5±0.2

31.6±0.3

27.1±0.4

21.8±0.4

16.5±0.6

-----------------------------------------------------------------------------------------------------------Diglycine

55.3±0.2

50.5±0.3

61

43.5±0.1

35.6±0.5

28.5±0.6

-----------------------------------------------------------------------------------------------------------Triglycine

67.3±0.3

60.1±0.3

50.5±0.1

39.5±0.2

30.2±0.3

-----------------------------------------------------------------------------------------------------------Tetraglycine

77.4±0.3

68.0±0.3

59.0±0.3

47.8±0.2

35.9±0.2

------------------------------------------------------------------------------------------------------------

Table 2-3. Partial Molar Volumes, V° (cm3 mol-1), of Solutes as a Function of GB Concentration ----------------------------------------------------------------------------------------------------------Compounds

0M

1M

2M

3M

4M

----------------------------------------------------------------------------------------------------------N-Ac-Gly-NH2

91.1±0.1

90.5±0.2

90.4±0.2

90.5±0.1

90.7±0.3

----------------------------------------------------------------------------------------------------------N-Ac-Ala-NH2

108.3±0.2 107.3±0.3 106.6±0.2 106.1±0.3 106.5±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Val-NH2

138.5±0.2 137.0±0.5 136.1±0.3 135.6±0.5 135.4±0.1

----------------------------------------------------------------------------------------------------------N-Ac-Leu-NH2

156.4±0.3 154.4±0.1 153.5±0.2 152.6±0.2 153.1±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Ile-NH2

153.8±0.3 152.3±0.2 151.4±0.3 149.9±0.4 149.2±0.2

----------------------------------------------------------------------------------------------------------N-Ac-Pro-NH2

126.1±0.4 125.2±0.5 124.4±0.3 123.9±0.2 123.5±0.1

-----------------------------------------------------------------------------------------------------------

62

N-Ac-Phe-NH2

170.7±0.2 168.6±0.1 166.7±0.4 166.6±0.3 168.0±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Trp-NH2

192.9±0.2 191.0±0.4 189.8±0.3 189.6±0.1 190.3±0.5

----------------------------------------------------------------------------------------------------------N-Ac-Met-NH2

153.6±0.1 152.9±0.4 152.4±0.3 152.1±0.2 151.9±0.3

----------------------------------------------------------------------------------------------------------N-Ac-Tyr-NH2

172.9±0.3 172.4±0.2 172.3±0.1 172.3±0.1 172.3±0.4

----------------------------------------------------------------------------------------------------------N-Ac-Gln-NH2

141.2±0.2 141.8±0.1 142.2±0.4 142.6±0.5 142.9±0.3

----------------------------------------------------------------------------------------------------------Glycine

43.5±0.2

44.3±0.2

45.3±0.2

46.4±0.1

47.2±0.3

-----------------------------------------------------------------------------------------------------------Diglycine

76.7±0.2

77.9±0.2

79.4±0.1

80.5±0.3

81.4±0.2

-----------------------------------------------------------------------------------------------------------Triglycine

112.7±0.3 113.9±0.3 115.4±0.1 117.5±0.1 119.4±0.4

-----------------------------------------------------------------------------------------------------------Tetraglycine

149.3±0.4 152.0±0.4 154.3±0.3 156.4±0.3 158.5±0.2

------------------------------------------------------------------------------------------------------------

63

Table 2-4. Partial Molar Adiabatic Compressibilities, K°S (10-4 cm3 mol-1 bar-1), of Solutes as a Function of GB Concentration ----------------------------------------------------------------------------------------------------------Compounds

0M

1M

2M

3M

4M

----------------------------------------------------------------------------------------------------------N-Ac-Gly-NH2

-0.8±0.2

3.7±0.4

8.2±0.4

11.9±0.4

14.9±0.3

----------------------------------------------------------------------------------------------------------N-Ac-Ala-NH2

-0.2±0.3

4.6±0.4

9.0±0.3

12.3±0.8

15.9±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Val-NH2

-0.7±0.3

5.1±0.7

10.9±0.4

16.4±0.6

20.7±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Leu-NH2

-0.5±0.4

6.8±0.6

13.6±0.3

20.1±0.4

25.9±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Ile-NH2

-2.7±0.4

3.6±0.4

9.6±0.7

14.3±0.5

17.9±0.8

----------------------------------------------------------------------------------------------------------N-Ac-Pro-NH2

-6.0±0.5

0.2±0.7

5.9±0.6

10.1±0.4

13.3±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Phe-NH2

-0.2±0.3

8.3±0.3

15.7±0.5

21.0±0.9

24.4±0.9

----------------------------------------------------------------------------------------------------------N-Ac-Trp-NH2

2.6±0.2

11.3±0.7

19.8±0.3

28.6±0.6

33.0±0.8

----------------------------------------------------------------------------------------------------------N-Ac-Met-NH2

-2.2±0.2

6.0±0.4

13.8±0.5

19.6±0.6

24.7±0.6

-----------------------------------------------------------------------------------------------------------

64

N-Ac-Tyr-NH2

5.9±0.4

13.4±0.2

21.7±0.2

28.2±0.3

33.5±0.6

----------------------------------------------------------------------------------------------------------N-Ac-Gln-NH2

-2.6±0.2

4.9±0.3

11.8±0.5

17.7±0.5

22.2±0.5

----------------------------------------------------------------------------------------------------------Glycine

-26.6±0.2

-19.2±0.4

-12.7±0.5

-6.7±0.4

-2.3±0.7

-----------------------------------------------------------------------------------------------------------Diglycine

-39.7±0.3

-29.8±0.4

-19.5±0.2

-10.9±0.6

-4.6±0.6

-----------------------------------------------------------------------------------------------------------Triglycine

-43.9±0.4

-31.1±0.5

-18.7±0.2

-8.6±0.2

0.6±0.4

-----------------------------------------------------------------------------------------------------------Tetraglycine

-46.0±0.5

-30.8±0.5

-16.5±0.4

-4.9±0.3

4.4±0.3

------------------------------------------------------------------------------------------------------------

Table 2-5. Partial Molar Volume Contributions of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), V (cm3 mol-1), as a Function of GB Concentration ----------------------------------------------------------------------------------------------------------Compounds

0M

1M

2M

3M

4M

----------------------------------------------------------------------------------------------------------Ala

17.3±0.2

16.8±0.3

16.1±0.3

15.6±0.4

15.9±0.5

----------------------------------------------------------------------------------------------------------Val

47.2±0.2

46.5±0.5

45.7±0.4

45.2±0.5

44.8±0.3

----------------------------------------------------------------------------------------------------------Leu

65.3±0.3

63.9±0.2

65

63.1±0.2

62.2±0.2

62.5±0.5

----------------------------------------------------------------------------------------------------------Ile

62.7±0.3

61.8±0.3

61.0±0.4

59.4±0.4

58.6±0.3

----------------------------------------------------------------------------------------------------------Pro

35.1±0.4

34.7±0.5

34.0±0.4

33.4±0.2

32.8±0.3

----------------------------------------------------------------------------------------------------------Phe

79.6±0.3

78.1±0.2

76.2±0.4

76.2±0.4

77.4±0.5

----------------------------------------------------------------------------------------------------------Trp

101.8±0.1 100.5±0.4 99.4±0.3

99.2±0.2

99.6±0.5

----------------------------------------------------------------------------------------------------------Met

62.5±0.2

62.4±0.4

62.0±0.3

61.7±0.2

61.2±0.4

----------------------------------------------------------------------------------------------------------Tyr

81.9±0.3

81.9±0.3

81.9±0.2

81.8±0.2

81.6±0.5

----------------------------------------------------------------------------------------------------------Gln

50.2±0.2

51.2±0.2

51.8±0.4

52.2±0.5

52.3±0.4

-----------------------------------------------------------------------------------------------------------CH2CONH-

36.6±0.5

38.1±0.5

38.9±0.3

39.0±0.3

39.1±0.4

------------------------------------------------------------------------------------------------------------

66

Table 2-6. Partial Molar Adiabatic Compressibility Contributions of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), KS (10-4 cm3 mol-1 bar-1), as a Function of GB Concentration ----------------------------------------------------------------------------------------------------------Compounds

0M

1M

2M

3M

4M

----------------------------------------------------------------------------------------------------------Ala

0.7±0.4

0.9±0.6

0.7±0.5

0.4±0.9

1.0±0.6

----------------------------------------------------------------------------------------------------------Val

0.1±0.3

1.4±0.8

2.6±0.5

4.4±0.8

5.8±0.7

----------------------------------------------------------------------------------------------------------Leu

0.3±0.5

3.1±0.8

5.3±0.5

8.2±0.6

11.1±0.7

----------------------------------------------------------------------------------------------------------Ile

-1.9±0.4

0.0±0.5

1.4±0.8

2.4±0.7

3.0±0.9

----------------------------------------------------------------------------------------------------------Pro

-5.2±0.5

-3.5±0.8

-2.3±0.7

-1.8±0.6

-1.6±0.6

----------------------------------------------------------------------------------------------------------Phe

0.7±0.4

4.7±0.5

7.5±0.6

9.1±1.0

9.5±0.9

----------------------------------------------------------------------------------------------------------Trp

3.4±0.3

2.0±0.8

11.6±0.5

16.6±0.7

18.1±0.8

----------------------------------------------------------------------------------------------------------Met

-1.3±0.3

2.3±0.6

5.5±0.6

7.7±0.8

9.8±0.7

----------------------------------------------------------------------------------------------------------Tyr

6.7±0.4

9.7±0.5

67

13.4±0.4

16.3±0.6

18.6±0.7

----------------------------------------------------------------------------------------------------------Gln

-1.7±0.3

1.2±0.5

3.5±0.6

5.8±0.6

7.3±0.6

-----------------------------------------------------------------------------------------------------------CH2CONH-

-2.2±0.6

0.3±0.7

2.2±0.4

3.7±0.4

3.8±0.5

------------------------------------------------------------------------------------------------------------

Analytical Treatment of Volumetric Data. We analyze the GB-dependences of the volume and compressibility contributions of the amino acid side chains and the glycyl unit within the framework of a recently developed statistical thermodynamic model.11,13 In this model, the binding of a cosolvent molecule to a hydrated solute is accompanied by a release of r water molecules from the hydration shell of a solute to the bulk. If there are n binding sites for the principal solvent (water), the maximum number of cosolvent binding sites is n/r. Under the simplification of n/r identical and independent cosolvent binding sites, a change in volume associated with the transfer of a solute from water to a concentrated cosolvent solution is given by the relationship

V° = VC - 1nV°1 + V(n/r)(a3/a1r)k / [1 + (a3/a1r)k]

11

:

(4)

where VC is the differential volume of a cavity enclosing a solute in a concentrated cosolvent solution and water; k is the effective equilibrium constant for the reaction in which a cosolvent molecule replaces r water molecules by binding to each of the n/r binding sites a1 and a3 are the activities of water and cosolvent, respectively; V = V0 + 1rV°1 - 3V°3 is the change in volume associated with replacement of water with

68

cosolvent normalized per binding site in a concentrated cosolvent solution; V0 is the solvent exchange volume in an ideal solution; V°1 and V°3 are the excess partial molar volumes of water and cosolvent in a concentrated solution, respectively and 1 and 3 are the correction factors reflecting the influence of the bulk solvent on the properties of solvating water and cosolvent, respectively. The values of 1 and 3 may change from 0 (the properties of the solvation shell change in parallel with those of the bulk) to 1 (the properties of the solvation shell are independent of those of the bulk).11,13 A change in isothermal compressibility accompanying the water-to-cosolvent transfer of a solute is described by the relationship

11

:

K°T = KTC - 1nK°T1 + KT(n/r)(a3/a1r)k / [1 + (a3/a1r)k] + V2(n/r)(a3/a1r)k / RT[1 + (a3/a1r)k]2

(5)

where KTC = -(VC/P)T is the differential compressibility of the cavity enclosing a solute in water and a concentrated cosolvent solution; KT = KT0 + 1rK°T1 - 3K°T3 is the change in compressibility associated with replacement of water with cosolvent normalized per binding site in a concentrated cosolvent solution; K°T1 and K°T3 are the excess partial molar isothermal compressibilities of water and cosolvent in a concentrated cosolvent solution; KT0 = -(V0/P)T is the change in compressibility associated with the solvent replacement in an ideal solution. Scaled particle theory (SPT)-based calculations have revealed that, for solutes with hard-sphere diameters smaller than ~6 Å (corresponding to the size of N-acetyl amino acid amides), the differential cavity volume, VC, in Eq. (4) does not exceed ~1 cm3mol-1

69

as the GB concentration changes from 0 to 4 M 24. Consequently, the VC term in Eq. (3) and, by extension, the KTC term in Eq. (5) can be neglected in the subsequent analysis. In Eqs. (4) and (5), the number of water binding sites, n, for each solute can be taken equal to the number of water molecules in direct contact with a solute (confined within the first coordination layer). The latter can be calculated as the ratio of the solvent-accessible surface area of a solute to 9 Å2, the effective cross-section of water molecule. The values of n for individual amino acid side chains and the glycyl unit are presented in our previous publication 11. The number of water molecules, r, replaced by GB in the vicinity of a solute can be estimated as the ratio of n to the maximum number of GB molecules that can be in simultaneous contact with a solute. The latter is equal to the number of GB molecule within the first solute-cosolvent coordination sphere. For a spherical solute with a radius rs, the number of water and GB molecules within their respective (solute-water and solute-cosolvent) first coordination spheres are equal to (rs + rW)2/rW2 and (rs + rGB)2/rGB2, respectively (rW and rGB are the hard sphere radii of water and GB, respectively). Hence, the value of r is given by [(rs/rW + 1)/( rs/rGB +1)]2, where rW = 1.4 Å and rGB = 3.04 Å are the hard sphere radii of water and GB. The latter was calculated based on the spherical approximation of a GB molecule as rGB = (3VGB/4)1/3, where VGB is the van der Waals volume of GB.24 For solutes with radii, rs, corresponding to the protein range (above ~5 Å), the number of water molecules, r, released to the bulk upon GB binding is ~4. Hence, in our analysis below we use the value of r equal to 4. The mole fraction activity of water, a1, and the molar activity of GB, a3, have been reported by Felitsky and Record.25 The values of a1 are 1.000, 0.976, 0.939, 0.880, and

70

0.782, while the values of a3 are 0, 1.62, 5.65, 16.59, and 52.61 at 0, 1, 2, 3, and 4 M, respectively.25 We use these values in Eqs. (4) and (5) when analyzing our data below. The relationship between a1 and a3 is well approximated by the exponential function a1 = 0.76 + 0.24exp(-a3/24.11). Eq. (5) has been derived for partial molar isothermal compressibility data.11,13 However, its use for treating the partial molar adiabatic compressibility data presented in Table 2-5 is warranted given the small difference between the partial molar adiabatic and isothermal compressibilities of solutes in aqueous solutions due to the large heat capacity and small expansibility of water.26,27 When applied to analysing cosolvent-induced changes in the partial molar adiabatic compressibility of a solute, K°S, the isothermal quantities K°T1, KT, and KT0 in Eq. (5) should be replaced by their adiabatic analogs. The experimental values of V°1 and K°S1 (shown in Table 2-1) can be related to the activity of GB, a3, via the functions; V°1(cm3mol-1) = 1.6510-3a3 - 1.1510-5a32 and K°S1(10-4 cm3mol-1bar-1) = -0.79 + 0.79exp(-a3/25.44). In Eqs. (4) and (5), the correction factor 3 can be taken to be 0, while 1 can be taken equal to the nonpolar fraction of the solvent accessible surface area of a solute or an individual atomic group.11,13 The specific values of 1 for the amino acid side chains and the glycyl unit have been estimated in a previous work.11 We used Eqs. (4) and (5) to fit the GB-dependences of the volume and compressibility contributions of the amino acid side chains and the glycyl unit presented in Tables 2-5 and 2-6 and to determine the effective binding constants, k, and changes in volume, V0, and adiabatic compressibility, KS0, associated with replacement of water with cosolvent at the binding site of a solute in an ideal solution. Figure 2-1 shows the representative

71

dependences of the volume (panel A) and adiabatic compressibility (panel B) contributions of the leucine side chain on the activity of GB, a3, approximated by Eqs. (4) and (5), respectively. Table 2-7 lists our determined values of k (columns 6 and 7), V0 (column 4), and KS0 (column 5) for the 10 amino acid side chains studied here, the glycyl residue, and the zwitterionic amino acid glycine as an example of a highly charged solute. Within the context of the analysis presented below, it is pertinent to note that the binding of GB to a solute may be accompanied by disruption of GB-water and GB-GB interactions. Clearly, in this case, the values of k, V0 and KS0 will be affected by dehydration of GB and disruption of GB-GB contacts, although the binding-induced disruption of pre-existing GB interactions is not formally included in the model.

72

(A)

66

64

3

V°, cm mol

-1

65

63

62

61

0

10

20

30

40

50

60

a 3, M

12

(B)

8

-1

KS°, 10 cm mol bar

-1

10

-4

3

6 4 2 0 -2

0

10

20

30

40

50

60

a3, M

Figure 2-1. The partial molar volume (panel A) and adiabatic compressibility (panel B) contributions of the leucine side chain as a function of GB activity. The experimental points were fitted using Eqs. (4) (panel A) and (5) (panel B).

73

Effective Binding Constants. Inspection of Table 2-7 reveals that the effective binding constants, k, determined from the volume (column 6) and compressibility (column 7) data may significantly differ from each other. Given the larger GB-induced changes in compressibility relative to error of measurements compared to those in volume, it is reasonable to expect that the values of k determined from the compressibility data are more reliable than those determined from the volume data. In fact, the ratio of the net compressibility change-to-error ratio is, on average, four times as large as the volume change-to-error ratio. Therefore, in our analysis below, we use compressibility-based estimates of the binding constants with the exception of Ala. For the Ala side chain, the GB-induced changes in compressibility are small which makes them unsuitable for any reliable estimate of k. For Ala, we use, therefore, the value of k derived from the partial molar volume measurements. The binding constants, k, range from 0.02 to 0.32 M-1. These values are comparable to those we have determined for the binding of urea, a denaturing cosolvent.11 This observation is significant and suggests that protein-GB interactions are not negligible. Thus, the stabilizing action of GB results not from the weakness of its interactions with protein groups but rather from the highly unfavourable free energy of cavity formation in a concentrated GB solution relative to that in water (see below).

74

Table 2-7. The Correction Factor, 1, the Number of Binding Sites for Water, n, Equilibrium Constants, k, and Changes in Volume, V0, and Adiabatic Compressibility, KS0, Accompanying the Binding of GB to Amino Acid Side Chains and the Glycyl Unit in an Ideal Solution -----------------------------------------------------------------------------------------------------------Side Chain

1a

na

∆V0 cm3 mol-1

∆KS0104

kb

cm3 mol-1 bar-1 M-1

kc M-1

-----------------------------------------------------------------------------------------------------------Ala

1.0

7

-0.93±0.09

N/A

0.33±0.15

N/A

-----------------------------------------------------------------------------------------------------------Val

1.0

13

-0.79±0.02

1.24±0.34

0.27±0.03

0.02±0.02

-----------------------------------------------------------------------------------------------------------Leu

1.0

16

-0.75±0.04

2.53±0.21

0.46±0.13

0.06±0.03

-----------------------------------------------------------------------------------------------------------Ile

1.0

16

-1.09±0.05

1.00±0.09

0.10±0.02

0.13±0.06

-----------------------------------------------------------------------------------------------------------Pro

1.0

12

-0.79±0.03

1.11±0.03

0.10±0.02

0.29±0.05

-----------------------------------------------------------------------------------------------------------Phe

1.0

19

-0.64±0.11

1.80±0.03

0.90±1.04

0.32±0.03

-----------------------------------------------------------------------------------------------------------Trp

0.9

23

-0.44±0.03

2.54±0.08

0.74±0.36

0.13±0.02

-----------------------------------------------------------------------------------------------------------Met

0.7

17

-0.32±0.02

2.50±0.12

75

0.04±0.01

0.19±0.05

-----------------------------------------------------------------------------------------------------------Tyr

0.8

20

0

2.30±0.09

N/A

0.12±0.03

-----------------------------------------------------------------------------------------------------------Gln

0.4

13

0.66±0.01

2.73±0.11

0.48±0.03

0.18±0.03

------------------------------------------------------------------------------------------------------------CH2CONH-

0.4

6

1.70±0.04

4.10±0.08

0.88±0.12

0.26±0.03

-----------------------------------------------------------------------------------------------------------Glycine

1.0

15

1.04±0.03

6.29±0.27

0.13±0.01

0.17±0.03

-----------------------------------------------------------------------------------------------------------a

From Ref. 11.

b

Calculated from volume data with Eq. (4).

c

Calculated from compressibility data with Eq. (5).

Interaction, Cavity, and Transfer Free Energies. The differential free energy of cavity formation, GC, in Eq. (1) can be evaluated within the framework of scaled particle theory (SPT).12,28,29 The free energy of cavity formation, GC, in a binary mixture is given by the relationship:

GC = RT[-ln(1 - 3) + 32dS/(1 - 3) + 31dS2/(1 - 3) + 4.522dS2/(1 - 3)2 + NAPdS3/(6RT)] (6)

where dS is the diameter of a solute molecule the hard-sphere pressure, P, is computed from P/(RT) = 60/(1 - 3) + 1812/(1 - 3)2 + 1823/(1 - 3)3; NA is Avogadro's number; k

76

m

= (NA/6)  Cidik; k has the values of 0, 1, 2, and 3; m is the number of solvent i 1

components; Ci and di are, respectively, the molar concentration and the molecular diameter of the i-th solvent component.12,28,30 We used Eq. (6) to calculate the free energies of cavity formation, GC, for our solutes in water and concentrated GB solutions. In these calculations, we used hard sphere diameters of water and GB of 2.76 and 6.08 Å, respectively. The hard sphere diameter of each solute was calculated from its van der Waals volume, VW, using the spherical approximation; dS = (6VW/)1/3. The van der Waals volume, VW, of each solute under study was calculated additively based on its chemical structure and the group contributions reported by Bondi.31 The GC contribution for a specific side chain was computed as the difference between the values of GC corresponding to the respective N-acetyl amino acid amide and N-acetyl glycine amide. The free energy of cavity formation of the glycyl unit was evaluated as the difference between the value of GC for diglycine and glycine. Table 2-8 presents the values of GC we evaluated for the amino acid side chains and the glycyl unit.

77

Table 2-8. Change in Free Energy of Cavity Formation, GC (kcal mol-1), of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), Calculated as a Function of GB Concentration Using Eq. (6) -----------------------------------------------------------------------------------------------------------Side Chain

0

1

2

3

4

-----------------------------------------------------------------------------------------------------------Ala

0

0.14

0.31

0.53

0.80

-----------------------------------------------------------------------------------------------------------Val

0

0.42

0.94

1.58

2.39

-----------------------------------------------------------------------------------------------------------Leu

0

0.57

1.25

2.10

3.18

-----------------------------------------------------------------------------------------------------------Ile

0

0.57

1.25

2.10

3.18

-----------------------------------------------------------------------------------------------------------Pro

0

0.32

0.72

1.22

1.84

-----------------------------------------------------------------------------------------------------------Phe

0

0.73

1.60

2.69

4.07

-----------------------------------------------------------------------------------------------------------Trp

0

0.95

2.11

3.55

5.37

-----------------------------------------------------------------------------------------------------------Met

0

0.57

1.26

2.13

3.22

-----------------------------------------------------------------------------------------------------------Tyr

0

0.80

1.76

78

2.96

4.48

-----------------------------------------------------------------------------------------------------------Gln

0

0.54

1.20

2.02

3.05

------------------------------------------------------------------------------------------------------------CH2CONH-

0

0.43

0.96

1.61

2.44

------------------------------------------------------------------------------------------------------------

Within the framework of the assumption of n/r identical and independent cosolvent binding sites on the solute, GI is linked to the effective binding constant, k, via the relationship:

GI = -(n/r)RT ln[(a1/a10)r+ k(a3/a10r)]

(7)

where a10 is the activity of water in the absence of cosolvent.11 At low concentrations of a solute, when a10  0, Eq. (7) simplifies to the form:

GI = -(n/r)RT ln(a1r+ ka3)

(8)

79

Table 2-9. Differential Free Energy of Interaction, GI (kcal mol-1), of Amino Acid Side Chains and Glycyl Unit (-CH2CONH-), Calculated as a Function of GB Concentration Using Eq. (8) -----------------------------------------------------------------------------------------------------------Side Chain

0

1

2

3

4

-----------------------------------------------------------------------------------------------------------Ala

0

-0.38

-1.01

-1.87

-2.99

-----------------------------------------------------------------------------------------------------------Val

0

0.12

0.22

0.14

-0.68

-----------------------------------------------------------------------------------------------------------Leu

0

-0.01

-0.26

-1.11

-2.99

-----------------------------------------------------------------------------------------------------------Ile

0

-0.26

-0.98

-2.40

-4.68

-----------------------------------------------------------------------------------------------------------Pro

0

-0.57

-1.57

-3.00

-4.88

-----------------------------------------------------------------------------------------------------------Phe

0

-1.00

-2.67

-5.00

-8.00

-----------------------------------------------------------------------------------------------------------Trp

0

-0.38

-1.41

-3.45

-6.73

-----------------------------------------------------------------------------------------------------------Met

0

-0.49

-1.55

-3.33

-5.89

-----------------------------------------------------------------------------------------------------------Tyr

0

-0.29

-1.11

80

-2.82

-5.63

-----------------------------------------------------------------------------------------------------------Gln

0

-0.35

-1.13

-2.46

-4.40

------------------------------------------------------------------------------------------------------------CH2CONH-

0

-0.25

-0.72

-1.41

-2.35

------------------------------------------------------------------------------------------------------------

Table 2-10. Changes in Free Energies, Gtr(exp) (kcal mol-1), Calculated for the Water-to-GB Transfer of Amino Acid Side Chains and the Glycyl Unit (-CH2CONH-) Using Eq. (1). -----------------------------------------------------------------------------------------------------------Side Chain

0

1

2

3

4

-----------------------------------------------------------------------------------------------------------Ala

0

-0.24

-0.69

-1.33

-2.18

-----------------------------------------------------------------------------------------------------------Val

0

0.55

1.17

1.72

1.71

-----------------------------------------------------------------------------------------------------------Leu

0

0.55

0.99

0.99

0.19

-----------------------------------------------------------------------------------------------------------Ile

0

0.30

0.27

-0.30

-1.50

-----------------------------------------------------------------------------------------------------------Pro

0

-0.24

-0.84

-1.78

-3.04

-----------------------------------------------------------------------------------------------------------Phe

0

-0.28

-1.07

81

-2.31

-3.94

-----------------------------------------------------------------------------------------------------------Trp

0

0.57

0.71

0.10

-1.35

-----------------------------------------------------------------------------------------------------------Met

0

0.08

-0.28

-1.20

-2.67

-----------------------------------------------------------------------------------------------------------Tyr

0

0.51

0.65

0.14

-1.15

-----------------------------------------------------------------------------------------------------------Gln

0

0.19

0.07

-0.44

-1.35

------------------------------------------------------------------------------------------------------------CH2CONH-

0

0.18

0.24

0.20

0.10

------------------------------------------------------------------------------------------------------------

Table 2-9 presents our calculated values of GI for the amino acid side chains and the glycyl unit at different GB concentrations. We used these results in conjunction with the data on the cavity contribution, GC, listed in Table 2-8 to compute the transfer free energies, Gtr, from Eq. (1). The results of these computations are tabulated in Table 2-10 and plotted in Figure 2-2. Inspection of Table 2-9 and Figure 2-2 reveals that the aromatic side chain of Phe displays the most favourable interactions with GB. This observation is in excellent agreement with the results of a recent work from the Record laboratory.32 Based on the determined GB-surface interaction potentials for various functional groups, these authors concluded that the aromatic carbon surface of the benzyl group of benzoate is characterized by the most favourable interactions with GB. 32 Furthermore, our calculated moderately positive values of Gtr for the glycyl unit are

82

consistent with the prediction of moderate exclusion of GB from the vicinity of the peptide backbone made by Capp et al.32

3 2

Gtr, kcal mol

-1

1 0 -1 Ala Val Leu Ile Pro Phe Trp Met Tyr Gln Glycyl

-2 -3 -4 -5 0

10

20

30

40

50

60

a3 , M

Figure 2-2. Water-to-GB transfer free energies of the amino acid side chains and the glycyl unit plotted as a function of GB activity.

It is instructive to compare the experimental transfer free energies, Gtr, with those calculated from Eqs. (1), (6), and (8). Experimental data exist only for the changes in free energy accompanying the transfer of some amino acid side chains and the glycyl unit from water to a 1 M GB solution.6,33,34 Table 2-11 compares the calculated and experimental transfer free energies, Gtr, at 1 M GB. Although of the same order, there are some quantitative and qualitative disagreements between the two data sets. Several

83

possible explanations can be put forward to account for the discrepancies. First, SPT-based computations of GC may be unreliable due to their critical dependence on the assumed hard sphere diameter of the cosolvent molecule.12 The latter is not easy to determine given the necessity to approximate a non-rigid, non-spherical molecule by a rigid sphere.12 In fact, seemingly insignificant changes in the hard sphere diameters of the solvent and cosolvent molecules may substantially alter the magnitude and even the sign of GC.12 Second, the absolute magnitude of the interaction free energy, GI, may have been under- or overestimated since the number of GB binding sites in our model was taken equal to the maximum number of GB molecules that can be in contact with a solute. However, the actual number of GB binding sites may well be smaller than the maximum value given by n/r. The approximation of identical and independent GB-binding sites with a single k, while providing an adequate fit for the volume and compressibility data, may compromise quantitative evaluation of GI. A more complete equation for GI is based on the assumption of independent but not identical binding sites:

nh / r

GI = -RT  ln(a1r+ kia3)

(9)

i 1

where ki is the binding constant for the i-th binding site. Determination of individual binding constants, ki, from experimental GB-dependences of volume and compressibility is not possible. Clearly, a further level of complexity is to introduce cooperativity to the binding by allowing the sites to interact with one another.

84

Table 2-11. Calculated, Gtr(calc) (cal mol-1), and Experimental, Gtr(exp) (cal mol-1), Free Energies for the Transfer of the Amino Acid Side Chains and the Glycyl Unit (-CH2CONH-) from Water to 1 M GB -----------------------------------------------------------------------------------------------------------Side Chain

Gtr(calc)

Gtr(exp)

-----------------------------------------------------------------------------------------------------------Ala

183.25a

-235

-----------------------------------------------------------------------------------------------------------Val

158.85a

545

-----------------------------------------------------------------------------------------------------------Leu

160.75a

553

-----------------------------------------------------------------------------------------------------------Ile

177.21a

300

-----------------------------------------------------------------------------------------------------------Pro

53.32a

-241

-----------------------------------------------------------------------------------------------------------Phe

65.55a

-276

-----------------------------------------------------------------------------------------------------------Trp

574

-----------------------------------------------------------------------------------------------------------Met

164.32a

82

-----------------------------------------------------------------------------------------------------------Tyr

508

85

-----------------------------------------------------------------------------------------------------------Gln

186.05a

193

------------------------------------------------------------------------------------------------------------CH2CONH-

65±3b

183

-----------------------------------------------------------------------------------------------------------a

From Ref. 6.

b

From Ref. 34.

Third, the number of waters released to the bulk upon GB binding, r, may differ from our estimate of 4. Finally, the experimental transfer free energy data may suffer from the concentration-dependent effects as has been discussed in a previous paper.11 Given the large disparities between the calculated and experimental data on Gtr, we focus below on the qualitative inferences that can be drawn from our data. Inspection of Figure 2-2 in conjunction with the data presented in Tables 2-8 and 2-9 reveals several important observations. First, the transfer free energy, Gtr, results from a fine balance between the large GC and GI contributions. Second, the range of the magnitudes and the shape of the GB-dependence of Gtr depend on the identity of a specific solute group. The Ala, Pro, and Phe side chains display favourable values of Gtr within the entire GB concentration range. The other side chains and the glycyl unit exhibit unfavourable Gtr at low GB and favourable Gtr at high GB. The interplay between GC and GI results in pronounced maxima in the GB-dependences of Gtr for the Val, Leu, Ile, Trp, Tyr, and Gln side chains as well as the glycyl unit. This observation is in qualitative agreement with the maxima in the TM-versus-GB concentration plots observed

86

by Bolen and co-workers for ribonuclease A and lysozyme within the 3 to 4 M GB range.35 In addition, this observation is consistent with the analysis of the behavior of GB in the vicinity of lacI HTH DNA binding domain presented by Felitsky and Record which predicts a reversal of the stabilizing effect of GB at high concentrations.25 These authors have determined that the local-bulk partition coefficient, Kp, of GB near the protein is smaller than 1 between 0 and ~4 M GB but intersects the unity line and becomes larger than 1 at higher cosolvent concentrations.25

Volumetric Parameters. As is seen from Table 2-7, the values of V0 are negative for predominantly nonpolar amino acid side chains, but show a tendency to become positive for the polar and charged entities - the glutamine side chain, the glycyl residue, and the zwitterionic amino acid glycine. Recall that V0 is the change in volume accompanying the binding of a cosolvent (urea or GB) to a particular solute or an atomic group in an ideal solution. As such, V0 is contributed by changes in thermal, VT, and interaction, VI, volumes.36,37 The thermal volume, VT, which mainly results from thermally-induced mutual solute-solvent vibrations, represents the effective void volume around a solute. Note that VT is proportional to the number of solute-water and cosolvent-water contacts both of which decreases upon the association of a solute and cosolvent molecules.37 Consequently, a change in thermal volume, VT, associated with solute-cosolvent binding event is invariably negative. The interaction volume, V I, which reflects solvent contraction in the vicinity of polar and charged groups, may increase or remain the same upon solute-cosolvent binding events which are accompanied by release of waters of solute and cosolvent hydration to the bulk.36 The values of VI will be

87

large and positive if the associating species interact via polar and charged groups, while being near zero if the contacting groups are predominantly nonpolar.36 The observation that, for charged and polar groups, the values of V0 are positive is consistent with a picture in which VI prevails over VT. On other hand, the highly negative values of V0 observed for GB association with nonpolar groups suggest that, for these groups, VT prevails over VI. This observation can be accounted for by proposing that the contacts between GB and nonpolar groups of a solute are mainly implemented via the amino methyl groups of the former. Given the bulkiness of the the amino terminus of GB, -N(CH3)+, and, hence, a low charge density around it, waters solvating the amino methyl groups are likely to display structural, volumetric, and thermodynamic properties similar to those displayed by waters solvating nonpolar substances. Therefore, we propose that release of waters solvating the amino methyl groups of GB to the bulk should have a near zero contribution to VI. In this scenario, V0 is dominated by a negative change in VT, in agreement with the observed tendency. Changes in compressibility, KS0, accompanying the binding of GB to the molecular entities studied in this work are all positive. This observation is in line with the fact that, since at 25 °C, water molecules solvating nonpolar, charged, and most of polar atomic groups are characterized by a lower partial molar compressibility relative to bulk water.26,38 Consequently, release of hydration water to the bulk upon the association of GB with solute groups should result in positive changes in compressibility. In the aggregate, individual values of V0 and KS0 determined for water-cosolvent exchange reflect a host of molecular events including a decrease in the number of

88

water-cosolvent and water-solute contacts, release of waters of solute and GB hydration to the bulk, and formation of specific solute-cosolvent contacts. In addition, each of these events may have a relaxation contribution to KS0.39 Quantitative interpretation of and differentiation between the volumetric effects of these events is not a simple matter. However, independent of the "success" or "failure" of such interpretations or their veracity, the values of V0 and KS0 represent a volumetric signature of solvent-cosolvent exchange in the vicinity of various solute groups and can be used as such for identification and characterization of the differential solvation of atomic groups in water and water-cosolvent mixtures.

2.6 CONCLUDING REMARKS We measured the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides and oligoglycines at GB concentrations ranging from 0 to 4 M. We used the resulting data to evaluate the volumetric contributions of the amino acid side chains and the glycyl unit (-CH2CONH-) as a function of GB concentration. The resulting data were analyzed within the framework of a statistical thermodynamic model to evaluate the equilibrium constant for the reaction in which a GB molecule binds each of the studied functionalities replacing four water molecules. We calculated the free energy of the transfer of functional groups from water to concentrated GB solutions, Gtr, as the sum of a change in the free energy of cavity formation, GC, and the differential free energy of solute-solvent interactions, GI, in a concentrated GB solution and water. Our data suggest that the transfer free energy, Gtr, results from a fine balance between the

89

large GC and GI contributions. The range of the magnitudes and the shape of the GB-dependence of Gtr depend on the identity of a specific solute group. In particular, the interplay between GC and GI results in pronounced maxima in the GB-dependences of Gtr for the Val, Leu, Ile, Trp, Tyr, and Gln side chains as well as the glycyl unit. This observation is in qualitative agreement with the maxima in the TM-versus-GB concentration plots observed for ribonuclease A and lysozyme within the 3 to 4 M GB range.35

2.7 ACKNOWLEDGEMENTS This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to T.V.C. Y.L.S. gratefully acknowledges his graduate support from the CIHR Protein Folding Training Program.

90

2.8 REFERENCES 1. Yancey, P. H. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J. Exp. Biol. 2005, 208, 2819-2830. 2. Timasheff, S. N. Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv. Protein Chem. 1998, 51, 355-432. 3. Timasheff, S. N. Protein hydration, thermodynamic binding, and preferential hydration. Biochemistry 2002, 41, 13473-13482. 4. Schellman, J. A. Protein stability in mixed solvents: a balance of contact interaction and excluded volume. Biophys. J. 2003, 85, 108-125. 5. Hong, J.; Capp, M. W.; Anderson, C. F.; Saecker, R. M.; Felitsky, D. J.; Anderson, M. W.; Record, M. T., Jr. Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability. Biochemistry 2004, 43, 14744-14758. 6. Auton, M.; Bolen, D. W.; Rosgen, J. Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides. Proteins Struct. Funct. Bioinform. 2008, 73, 802-813. 7. Kumar, R. Role of naturally occurring osmolytes in protein folding and stability. Arch. Biochem. Biophys. 2009, 491, 1-6.

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8. Yancey, P. H.; Clark, M. E.; Hand, S. C.; Bowlus, R. D.; Somero, G. N. Living with water stress: evolution of osmolyte systems. Science 1982, 217, 1214-1222. 9. Auton, M.; Bolen, D. W. Application of the transfer model to understand how naturally occuring osmolytes affect protein stability. Osmosens. Osmosignal. 2007, 428, 397-418. 10. Davis-Searles, P. R.; Saunders, A. J.; Erie, D. A.; Winzor, D. J.; Pielak, G. J. Interpreting the effects of small uncharged solutes on protein-folding equilibria. Annu. Rev. Biophys. Biomolecul. Struct. 2001, 30, 271-306. 11. Lee, S.; Shek, Y. L.; Chalikian, T. V. Urea interactions with protein groups: a volumetric study. Biopolymers 2010, 93, 866-879. 12. Tang, K. E. S.; Bloomfield, V. A. Excluded volume in solvation: sensitivity of scaled-particle theory to solvent size and density. Biophys. J. 2000, 79, 2222-2234. 13. Lee, S. Y.; Chalikian, T. V. Volumetric properties of solvation in binary solvents. J. Phys. Chem. B 2009, 113, 2443-2450. 14. Eggers, F.; Funck, T. Ultrasonic measurements with milliliter liquid samples in 0.5-100 MHz range. Rev. Sci. Instrum. 1973, 44, 969-977. 15. Sarvazyan, A. P. Development of methods of precise ultrasonic measurements in small volumes of liquids. Ultrasonics 1982, 20, 151-154. 16. Eggers, F. Ultrasonic velocity and attenuation measurements in liquids with resonators, extending the MHz frequency range. Acustica 1992, 76, 231-240.

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17. Eggers, F.; Kaatze, U. Broad-band ultrasonic measurement techniques for liquids. Meas. Sci. Technol. 1996, 7, 1-19. 18. Sarvazyan, A. P.; Selkov, E. E.; Chalikyan, T. V. Constant-path acoustic interferometer with transition layers for precision measurements in small liquid volumes. Sov. Phys. Acoust. 1988, 34, 631-634. 19. Sarvazyan, A. P.; Chalikian, T. V. Theoretical analysis of an ultrasonic interferometer for precise measurements at high pressures. Ultrasonics 1991, 29, 119-124. 20. Hedwig, G. R.; Reading, J. F.; Lilley, T. H. Aqueous solutions containing amino acids and peptides .27. Partial molar heat capacities and partial polar volumes of some N-acetyl amino acid amides, some N-acetyl peptide amides and two peptides at 25 °C. J. Chem. Soc. Faraday Trans. 1991, 87, 1751-1758. 21. Hakin, A. W.; Hedwig, G. R. The partial molar heat capacities and volumes of some N-acetyl amino acid amides in aqueous solution over the temperature range 288.15 to 328.15 K. Phys. Chem. Chem. Phys. 2000, 2, 1795-1802. 22. Liu, J. L.; Hakin, A. W.; Hedwig, G. R. Amino acid derivatives as protein side-chain model compounds: the partial molar volumes and heat capacities of some N-acetyl-N'-methyl amino acid amides in aqueous solution. J. Solution Chem. 2001, 30, 861-883. 23. Hedwig, G. R.; Hoiland, H. Partial molar isentropic and isothermal compressibilities of some N-acetyl amino acid amides in aqueous solution at 298.15 K. Phys. Chem. Chem. Phys. 2004, 6, 2440-2445.

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24. Chalikian, T. V. Volumetric measurements in binary solvents: theory to experiment. Biophys. Chem. 2011, 156, 3-12. 25. Felitsky, D. J.; Record, M. T. Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface. Biochemistry 2004, 43, 9276-9288. 26. Chalikian, T. V.; Sarvazyan, A. P.; Breslauer, K. J. Hydration and partial compressibility of biological compounds. Biophys. Chem. 1994, 51, 89-107. 27. Blandamer, M. J.; Davis, M. I.; Douheret, G.; Reis, J. C. R. Apparent molar isentropic compressions and expansions of solutions. Chem. Soc. Rev. 2001, 30, 8-15. 28. Pierotti, R. A. Scaled particle theory of aqueous and non-aqueous solutions. Chem. Rev. 1976, 76, 717-726. 29. Desrosiers, N.; Desnoyers, J. E. Enthalpies, heat capacities, and volumes of transfer of tetrabutylammonium ion from water to aqueous mixed solvents from point of view of scaled particle theory. Can. J. Chem. 1976, 54, 3800-3808. 30. Lebowitz, J. L.; Helfand, E.; Praestga E. Scaled particle theory of fluid mixtures. J. Chem. Phys. 1965, 43, 774-779. 31. Bondi, A. Van der Waals volumes and radii. J. Phys. Chem. 1964, 68, 441-451. 32. Capp, M. W.; Pegram, L. M.; Saecker, R. M.; Kratz, M.; Riccardi, D.; Wendorff, T.; Cannon, J. G.; Record, M. T. Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values. Biochemistry 2009, 48, 10372-10379.

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33. Auton, M.; Bolen, D. W. Additive transfer free energies of the peptide backbone unit that are independent of the model compound and the choice of concentration scale. Biochemistry 2004, 43, 1329-1342. 34. Street, T. O.; Bolen, D. W.; Rose, G. D. A molecular mechanism for osmolyte-induced protein stability. Proc. Natl. Acad. Sci. U. S. A 2006, 103, 13997-14002. 35. Santoro, M. M.; Liu, Y. F.; Khan, S. M. A.; Hou, L. X.; Bolen, D. W. Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry 1992, 31, 5278-5283. 36. Kharakoz, D. P. Partial molar volumes of molecules of arbitrary shape and the effect of hydrogen bonding with water. J. Solution Chem. 1992, 21, 569-595. 37. Chalikian, T. V.; Filfil, R. How large are the volume changes accompanying protein transitions and binding? Biophys. Chem. 2003, 104, 489-499. 38. Kharakoz, D. P. Volumetric properties of proteins and their analogs in diluted water solutions .2. Partial adiabatic compressibilities of amino acids at 15-70 °C. J. Phys. Chem. 1991, 95, 5634-5642. 39. Chalikian, T. V. On the molecular origins of volumetric data. J. Phys. Chem. B 2008, 112, 911-917.

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CHAPTER 3: Glycine Betaine Interactions with Proteins: Insights from Volume and Compressibility Measurements

“Reprinted from Biochemistry, 52, Yuen Lai Shek & Tigran V. Chalikian,. Interactions of glycine betaine with proteins: insights from volume and compressibility measurements, 672-680. Copyright (2013), with permission from American Chemical Society”. Author‘s Contribution: Yuen Lai Shek performed all experiments, analyzed the data, and participated in the preparation of the manuscript.

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3.1 ABSTRACT We report the first application of volume and compressibility measurements to characterization of interactions between cosolvents (osmolytes) and globular proteins. Specifically, we measure the partial molar volumes and adiabatic compressibilities of cytochrome c, ribonuclease A, lysozyme, and ovalbumin in aqueous solutions of the stabilizing osmolyte glycine betaine (GB) at concentrations between 0 and 4 M. The fact that globular proteins do not undergo any conformational transitions in the presence of GB provides an opportunity to study the interactions of GB with proteins in their native states within the entire range of experimentally accessible GB concentrations. We analyze our resulting volumetric data within the framework of a statistical thermodynamic model in which each instance of GB interaction with a protein is viewed as a binding reaction that is accompanied by release of four water molecules. From this analysis, we calculate the association constants, k, as well as changes in volume, V0, and adiabatic compressibility, KS0, accompanying each GB-protein association event in an ideal solution. By comparing these parameters with the similar characteristics (k, V0, and KS0) determined for low molecular weight analogs of proteins, we conclude that there are no significant cooperative effects involved in GB interactions with any of the proteins studied in this work. We also evaluate the free energies of direct GB-protein interactions. The energetic properties of GB-protein association appear to scale with the size of the protein. For all proteins, the highly favourable change in free energy associated with direct protein-cosolvent interactions is nearly compensated by an unfavourable free energy of cavity formation (excluded volume effect) to yield a modestly unfavourable free energy of the transfer of a protein from water to a GB-water mixture. 97

3.2 INTRODUCTION

Water-soluble cosolvents may exert a profound influence on protein stability by selectively shifting the conformational equilibrium between the protein species towards the native or denatured state (1-4). In many instances, cosolvent-induced modulation of protein stability is of biological relevance by facilitating survival of cells at extreme conditions (5-8). The mode of action of water-soluble cosolvents on the stability and biological activity of proteins remains a much debated topic in biophysical chemistry although new insights increasingly emerge from a combination of experimental and theoretical studies (1;3;4;9-15). In one approach, the net free energy of interactions between cosolvent and a protein is analyzed as the sum of the contribution arising from direct cosolvent-protein interactions and the contribution reflecting the differential free energy of cavity formation in pure water and a water-cosolvent mixture (excluded volume effect) (9;16). While the differential effect of cavity formation can be evaluated, in principle, within the framework of scaled particle theory (SPT) or some other more rigorous computational approach, it is a challenge to detect and quantify thermodynamically direct protein-cosolvent interactions. To address this deficiency, we have begun a program in which volumetric measurements are systematically employed in conjunction with a statistical thermodynamic algorithm to characterize interactions of various cosolvents with proteins and individual protein groups (17-20). In a recent paper, we have used volumetric measurements to study the interactions of the stabilizing osmolyte glycine betaine (GB) with low molecular weight compounds modeling proteins (20). We have determined the association constants, k, and changes in volume, V0, and adiabatic compressibility, KS0, accompanying the binding of GB to 98

various atomic groups in low molecular weight model compounds (20). In the present work, we extent the volumetric approach to characterization of the interactions of GB with globular proteins. The fact that globular proteins do not undergo any conformational transitions in the presence of GB provides one with the opportunity to study the interactions of GB with proteins in their native states within the entire range of experimentally accessible GB concentrations. One objective of this work is to understand if there are sizeable cooperative (nonadditive) effects involved in the interactions of GB with globular proteins which are absent in small molecules. This is a question of both fundamental and practical importance. The practical implications stem from the fact that low molecular weight compound data have been used extensively to develop additive models for evaluating the stabilizing and destabilizing effects of cosolvents on the conformational stability of proteins (14;21-23). To assess the cooperativity of GB interactions with proteins, we measure the partial molar volumes, V°, and adiabatic compressibilities, K°S, of four typical globular proteins differing in size and structural properties at GB concentrations ranging from 0 to 4 M. Specifically, the proteins we study are cytochrome c (12. 4 kDa), ribonuclease A (13.6 kDa), hen egg lysozyme (14.3 kDa), and ovalbumin (46.0 kDa). We analyze the resulting volumetric data within the framework of a statistical thermodynamic formalism and determine the average values of k, V0, and KS0. Comparison of these data with similar data previously obtained on small molecules enables one to judge about the presence of nonadditive effects in GB-protein interactions. Further, we use our association constants, k, to evaluate the free energy contributions of direct GB-protein interactions. These

99

results provide insights into the balance of forces governing the mode of action of individual cosolvents.

3.3 MATERIALS AND METHODS Materials. GB and the proteins hen egg lysozyme, ribonuclease A from bovine pancreas, ferricytochrome c from horse heart, and ovalbumin from chicken egg white were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, Ontario, Canada). All the reagents were of the highest purity commercially available. GB was used without further purification. To eliminate salts and any other low molecular weight compounds that may be present in the samples, the proteins were exhaustively dialyzed against distilled water and lyophilized.

Solution Preparation. Aqueous solutions of GB with concentrations between 0 and 4 M were prepared by weighing 10 to 50 g of GB and adding pre-estimated amounts of water to achieve the desired molalities, m. The molar concentrations, C, of a GB solution were determined from the molal values, m, using C = [1/(mW) + V/1000]-1, where W is the density of water and V is the apparent molar volume of GB. GB solutions were used as solvents for the proteins. Protein solutions were prepared by dissolving 5 to 10 mg of a solute material in a known amount of solvent (GB solution).

Spectroscopic Measurements. The concentrations of the proteins at each experimental GB concentration were determined spectrophotometrically at 25 °C with a Cary 300 Bio spectrophotometer (Varian Canada, Inc., Mississauga, Ontario, Canada).

100

The extinction coefficients for the proteins were determined individually for each GB concentration by dry-weight analysis. Table 3-S1 of Supporting Information lists our determined extinction coefficients for cytochrome c, ribonuclease A, lysozyme, and ovalbumin at 0, 1, 2, 3, and 4 M GB.

Near and far UV circular dichroism (CD) spectra of the proteins in the presence and absence of GB were recorded at 25 °C in a 1 mm pathlength cuvette using an Aviv model 62 DS spectropolarimeter (Aviv Associates, Lakewood, NJ). The proteins' concentrations were ~0.25 and ~1 mg/mL for the far and near UV CD measurements, respectively.

Determination of the Partial Molar Volumes and Adiabatic Compressibilities of the Proteins. Solution densities were measured at 25 °C with a precision of ±1.510-4 % using a vibrating tube densimeter (DMA-5000, Anton Paar, Gratz, Austria). The apparent molar volumes, V, of the proteins were evaluated from the relationship:

V = M/0 - ( - 0)/(0C)

(1)

where M is the molecular weight of the solute; C is the molar concentration of the solute;  and 0 are the densities of the solution and the solvent (water or a GB solution), respectively. Solution sound velocities, U, were measured at 25 °C at a frequency of 7.2 MHz using the resonator method and a previously described differential technique (24-27). The analysis of the frequency characteristics of the ultrasonic resonator cells required for

101

sound velocity measurements was carried out by a Hewlett Packard model E5100A network/spectrum analyzer (Mississauga, ON, Canada). For the type of ultrasonic resonators used in this work, the precision of sound velocity measurements is on the order of ±110-4 % (25;28;29). The key acoustic characteristics of a solute which can be derived directly from ultrasonic measurements is the relative molar sound velocity increment, [U] = (U - U0)/(U0C), where C is the molar concentration of a solute, and U and U0 are the sound velocities in the solution and the solvent, respectively. The values of [U] were combined with V calculate the apparent molar adiabatic compressibility, KS (30;31):

KS = S0 (2V - 2[U] - M/0)

(2)

where S0 = 0-1U0-2 is the coefficient of adiabatic compressibility of the solvent. The values of 0, U0, and S0 for GB solutions have been measured and reported previously (20). For each evaluation of V or KS, three to five independent measurements were carried out within a protein concentration range of 1 - 2 mg/mL. Our reported values of V or KS represent the averages of these measurements, while the errors were calculated as standard deviations. Given the small protein concentrations used in our study and the weak concentration dependences of the apparent molar volumes and adiabatic compressibilities of proteins (32;33), we do not discriminate below between the apparent and partial molar volumetric characteristics of the proteins.

102

3.4 RESULTS CD Measurements. To ensure that, in the presence of GB, the proteins do not undergo any structural alterations, we recorded the near (250 to 330 nm) and far (200 to 250 nm) UV CD spectra of the proteins in the presence and absence of GB (data not shown). No significant GB-induced changes in the CD spectra of any of the proteins were observed. We conclude that the proteins retain their native structures within the entire range of GB concentrations of 0 to 4 M used in this work. Consequently, we assume that our measured GB-dependent changes in the volumetric properties of the proteins can be ascribed predominantly to changes in their solvation.

Volumetric Measurements. Tables 3-1 and 3-2 list, respectively, the partial molar volumes, V°, and partial molar adiabatic compressibilities, K°S, of the proteins at various GB concentrations. The data on the relative molar sound velocity increments, [U], are included in Table 3-S2 of Supporting Information. Figures 3-1 and 3-2 graphically illustrate the data listed in Tables 3-1 and 3-2. Figure 3-1 presents the partial molar volumes of cytochrome c (panel A), ribonuclease A (panel B), lysozyme (panel C), and ovalbumin (panel D) plotted versus the activity of GB. The activities of GB, a3, for the concentration range of 0 to 4 M were taken from the literature (10). Inspection of Figure 3-1 reveals that, for all proteins studied in this work, the partial molar volumes increase hyperbolically with an increase in GB activity and level off at ~20 M.

103

Table 3-1. The Partial Molar Volumes of Proteins, V° (cm3 mol-1), of the Proteins at Various GB Concentrations --------------------------------------------------------------------------------------------------------------------[GB], M

Cytochrome c

Ribonuclease A

Lysozyme

Ovalbumin

--------------------------------------------------------------------------------------------------------------------0

9080±10

9620±10

10171±20

33390±50

--------------------------------------------------------------------------------------------------------------------0.5

9100±20

9640±10

10180±20

33440±20

--------------------------------------------------------------------------------------------------------------------1.0

9110±10

9670±20

10200±20

33500±20

--------------------------------------------------------------------------------------------------------------------1.5

9130±20

9680±20

10210±10

33550±20

--------------------------------------------------------------------------------------------------------------------2.0

9130±10

9690±10

10230±10

33580±20

--------------------------------------------------------------------------------------------------------------------2.5

9150±30

9700±20

10240±20

33640±50

--------------------------------------------------------------------------------------------------------------------3.0

9160±10

9710±20

10250±20

33670±10

--------------------------------------------------------------------------------------------------------------------3.5

9160±10

9720±10

10250±40

33690±40

--------------------------------------------------------------------------------------------------------------------4.0

9170±10

9720±10

10250±40

33710±10

---------------------------------------------------------------------------------------------------------------------

104

Table 3-2. The Partial Molar Adiabatic Compressibilities, K S ° (10-4 cm3 mol-1 bar-1), of the Proteins at Various GB Concentrations --------------------------------------------------------------------------------------------------------------------[GB], M

Cytochrome c

Ribonuclease A

Lysozyme

Ovalbumin

--------------------------------------------------------------------------------------------------------------------0

260±20

20±20

400±30

2790±80

--------------------------------------------------------------------------------------------------------------------0.5

360±30

100±20

500±30

3000±70

--------------------------------------------------------------------------------------------------------------------1.0

440±10

230±60

620±50

3230±50

--------------------------------------------------------------------------------------------------------------------1.5

520±30

340±20

720±10

3510±60

--------------------------------------------------------------------------------------------------------------------2.0

580±40

440±10

800±50

3720±40

--------------------------------------------------------------------------------------------------------------------2.5

664±40

550±80

900±50

3930±80

--------------------------------------------------------------------------------------------------------------------3.0

730±10

590±40

970±40

4120±40

--------------------------------------------------------------------------------------------------------------------3.5

770±50

630±60

1000±50

4210±70

--------------------------------------------------------------------------------------------------------------------4.0

800±40

650±50

1030±60

4240±40

---------------------------------------------------------------------------------------------------------------------

105

Figure 3-2 presents the GB-dependences of the partial molar adiabatic compressibilities of cytochrome c (panel A), ribonuclease A (panel B), lysozyme (panel C), and ovalbumin (panel D). Note that, analogous to volume, the partial molar adiabatic compressibilities of the proteins increase hyperbolically with an increase in the GB activity and level off at ~20 M.

106

9200

(a)

9175

3

V°, cm mol

-1

9150

9125

9100

9075

9050

0

10

20

30

40

50

60

40

50

60

a3, M

9750

(b)

9725

3

V°, cm mol

-1

9700

9675

9650

9625

9600

0

10

20

30

a3, M

107

10325

(c)

10300

-1

10225

V°, cm mol

10250

3

10275

10200 10175 10150 10125

0

10

20

30

40

50

60

a3, M 33800 33750

(d)

33700

3

V°, cm mol

-1

33650 33600 33550 33500 33450 33400 33350 33300

0

10

20

30

40

50

60

a3, M

Figure 3-1. The partial molar volumes of cytochrome c (a), ribonuclease A (b), lysozyme (c), and ovalbumin (d) as a function of GB activity.

108

900

(a)

-1

600

3

700

500

-4

KS°, 10 cm mol bar

-1

800

400 300 200 100

0

10

20

30

40

50

60

40

50

60

a 3, M 800

(b)

-1

500

3

600

400

-4

KS°, 10 cm mol bar

-1

700

300 200 100 0 -100

0

10

20

30

a3, M

109

1200

(c)

1100

900

-1

KS°, 10 cm mol bar

-1

1000

3

800

-4

700 600 500 400 300

0

10

20

30

40

50

60

40

50

60

a 3, M

4500

(d)

-1

3750

3

4000

3500

-4

KS°, 10 cm mol bar

-1

4250

3250 3000 2750 2500

0

10

20

30

a3, M

Figure 3-2. The partial molar adiabatic compressibilities of cytochrome c (a), ribonuclease A (b), lysozyme (c), and ovalbumin (d) as a function of GB activity.

110

Analysis of Partial Molar Volume Data. We analyzed the experimental data in Figures 3-1 and 3-2 within the framework of a statistical thermodynamic model in which the association of a cosolvent with a hydrated solute is accompanied by release of r water molecules from the hydration shell of the solute to the bulk (17;18;20). Four water molecules become released to the bulk per each GB-protein association event (r = 4) (20). If a solute has n binding sites for water, n/r is the maximum number of cosolvent binding sites. Assuming n/r identical and independent cosolvent binding sites, a change in volume associated with the transfer of a solute from water to a concentrated cosolvent solution has been expressed via the relationship (18):

V° = VC - 1nV°1 + V(n/r)(a3/a1r)k / [1 + (a3/a1r)k]

(3)

where a1 and a3 are the mole fraction activity of water and molar activity of GB, respectively; VC is the differential volume of the cavity enclosing a solute in a concentrated cosolvent solution and water (VC in a cosolvent solution minus VC in water); k is the effective equilibrium constant for the reaction in which a cosolvent molecule replaces r water molecules by binding to each of the n/r binding sites (SW r + C  SC + rW, where S, C, and W for the binding site, cosolvent, and water) V = V0 + 1rV°1 - 3V°3 is the change in volume associated with replacement of water with cosolvent normalized per binding site in a concentrated cosolvent solution; V0 is the solvent exchange volume in an ideal solution; V°1 and V°3 are the excess partial molar volumes of water and cosolvent in a concentrated solution and 1 and 3 are the correction factors reflecting the

111

influence of the bulk solvent on the properties of solvating water and cosolvent, respectively. Activities a1 and a3 have been determined as a function of GB concentration by Felitsky and Record (10). The values of 1 and 3 may change from 0 (the properties of the solvation shell change in parallel with those of the bulk) to 1 (the properties of the solvation shell are independent of those of the bulk). We used Eq. (3) to approximate the experimental data shown in Figures 3-1a, b, c, and d. We have reported the excess partial molar volume of water, V°1, as a function of GB concentration (20). The value of n was calculated for each protein as the ratio of its solvent accessible surface area, SA, to 9 Å2, the effective cross-section of a water molecule. Correction factors 1 and 3 in Eq. (3) can be evaluated for each protein based on the following considerations (17;18). Water molecules solvating charged atomic groups interact with the latter via strong charge-dipole interactions, thereby becoming highly compressed and partially immobilized. Given the small size of water molecules (and, hence, essentially localized interactions), waters influenced by charged groups should be relatively insensitive to the properties of water in the bulk. Thus, we have proposed that, for charged groups, 1 can be approximated by 1 (17;18). On a similar note, at low to moderate temperatures, waters solvating nonpolar groups become highly oriented in an attempt to maximize their mutual hydrogen bonds within a restricted configurational space. It is, therefore, reasonable to assume that the structural and thermodynamic properties of such waters should also be relatively insensitive to changes in the properties of bulk water. Thus, we have proposed that nonpolar groups are also characterized by a

112

1 close to 1. In contrast, waters hydrating polar (but uncharged) groups form continuous networks of hydrogen bonds extending from solute to water in the bulk and, therefore, should be significantly influenced by the latter. Consequently, we posit that, for polar groups, 1  0 (17;18). GB, like most water-soluble organic cosolvents, is bulkier than water and can form numerous hydrogen bonding and electrostatic interactions with its neighboring solvent and cosolvent molecules. Consequently, despite its being engaged in solute-solvent interactions, GB can still develop numerous interactions with solvent in the bulk thereby being influenced significantly by the latter. We, therefore, use in our analysis an approximation of 3  0 (17;18). On the basis of the discussion presented above, the correction factor 1 is roughly equal to the fraction of nonpolar and charged groups in the solvent accessible surface area of a solute [(Sn + SC)/SA], while 3 is effectively 0. The solvent accessible surface area of nonpolar, Sn, and charged, SC, groups and the total solvent accessible surface area, SA, were taken from a previous work (34). Our SPT-based computations has revealed that, for low molecular weight compounds (with hard sphere diameters on the order or less than 10 Å), the cavity contribution, VC, is small and does not exceed ~4 cm3 mol-1 at 4 M GB (19). However, for solutes as large as proteins, VC in Eq. (3) is not negligible. In principle, the values of VC can be calculated as a function of GB concentration using SPT (35;36). Our computations (data not shown) suggest that, for a solute with a diameter of 30 Å (which corresponds to a protein of the size of lysozyme), the dependence of VC on the concentration of GB is nearly parabolic; it decreases upon an increase in [GB] from 0 to ~1.2 M reaching a

113

minimum of -8 cm3 mol-1, then increases upon a further increase in [GB] passing zero at ~2.4 M and reaching the value of 35 cm3 mol-1 at 4 M GB. It should be noted, however, that the results of SPT-based calculations critically depend on the choice of the hard sphere diameters of the primary solvent and cosolvent molecules as well as on the precise values of their concentrations (37). In general, the problem of approximation of a complex molecular shape by a hard sphere does not have any universal solution with different approaches yielding significantly different estimates for the effective hard-sphere diameters of solvent and cosolvent molecules (37). Ambiguities exist not only with respect to solutes and water-miscible cosolvents, such as glycine betaine, but also with respect to the size of the water molecule itself (38). The impact of these effects on the validity of SPT-based calculations grows as the size of a solute increases, becoming very significant for solutes approaching the size of proteins. There is one more consideration that may limit the usefulness of SPT-based calculations for accurate volumetric analyses. SPT assumes a similar distribution of solvent and cosolvent molecules in the vicinity of the solute and in the bulk. However, depending on the nature of the cosolvent, the microenvironment of a solute, as reflected in the distribution of solvent and cosolvent molecules in its vicinity, may significantly differ from that in the bulk. The disparity between the local and bulk solvent properties may cause an additional deviation of the SPT-calculated characteristics of a solute. Considering these complexities and the errors associated with them, we do not attempt to evaluate the VC term in Eq. (3). Instead, we fit the experimental data shown in Figures 3-1 a, b, c, and d by Eq. (3) without explicitly taking into account the VC term. In

114

such a treatment, the cosolvent-induced change in the cavity volume, VC, will appear as an added contribution to the values of V0 determined from the fit.

Analysis of Partial Molar Compressibility Data. A change in isothermal compressibility accompanying the water-to-cosolvent transfer of a solute is described by the relationship (18):

K°T = KTC - 1nK°T1 + KT(n/r)(a3/a1r)k / [1 + (a3/a1r)k] + V2(n/r)(a3/a1r)k / RT[1 + (a3/a1r)k]2

(4)

where KTC = -(VC/P)T is the differential compressibility of the cavity enclosing a solute in water and a concentrated cosolvent solution; KT = KT0 + 1rK°T1 - 3K°T3 is the change in compressibility associated with replacement of water with cosolvent normalized per binding site in a concentrated cosolvent solution; K°T1 and K°T3 are the excess partial molar isothermal compressibilities of water and cosolvent in a concentrated cosolvent solution; KT0 = -(V0/P)T is the change in compressibility associated with solvent replacement in an ideal solution. Given the large heat capacity and small expansibility of water-based solutions, the difference between the partial molar adiabatic and isothermal compressibilities of a solute is small and, therefore, can be ignored. Thus, we use Eq. (4) to fit the experimental data shown in Figures 3-2a, b, c, and d (39). Consequently, instead of the excess partial molar isothermal compressibility of water, K°T1, we use, in Eq. (4), the excess partial molar

115

adiabatic compressibility, K°S1, that has been measured as a function of GB concentration (20). Analogous to volume, when analyzing our compressibility results, we ignore the differential cavity contribution KTC in Eq. (4). It should be emphasized that, in semiempirical analyses, the cavity contribution, KTC or KSC, is approximated reasonably well by the intrinsic compressibility, KM, of a solute (40-43). As discussed above, our CD spectral measurements did not reveal any significant GB-induced structural changes for the four proteins studied in this work. By extension, their intrinsic compressibilities should not be affected significantly by GB.

3.5 DISCUSSION Properties of GB-Protein Association. Table 3-3 presents the values of V0 and KS0 and the association constants, k, obtained from fitting our volume (Figure 3-1) and compressibility (Figure 3-2) data by Eqs. (3) and (4), respectively. Inspection of data in Table 3-3 reveals that, with the exception of ribonuclease A, there is good agreement between the equilibrium constants, k, derived from the volume and compressibility data. The disparity between the volume- and compressibility-based evaluations of the affinity constant, k, for ribonuclease A may reflect the large relative error of its individual partial molar volume data (see Figure 3-1c). In addition, the effect of unaccounted cavity volume, VC, on the value of k for ribonuclease A may be larger than that for the other proteins due to the structural and surface features of the former. In fact, since the relative error of the volume experimental points are generally larger than that of the compressibility points

116

(see Figures 3-1 and 3-2), we use below the values of k obtained from our compressibility data.

Table 3-3. Protein Solvent Accessible Surface Areas, SA (Å2), Correction 1, Changes in Volume, V0 (cm3 mol-1), and Adiabatic Compressibility, KS0 (10-4 cm3 mol-1bar-1), for GB-Protein Association in an Ideal solution, and equilibrium constants Determined from Volume, k(vol) (M-1) and Compressibility, k(comp) (M-1), Data --------------------------------------------------------------------------------------------------------------------Proteins

SA

1

V0

KS0

k(vol)

k(comp)

--------------------------------------------------------------------------------------------------------------------Cytochrome c

6115

0.75

0.509±0.007

2.97±0.04

0.26±0.02 0.24±0.02

--------------------------------------------------------------------------------------------------------------------Ribonuclease A

6790

0.63

0.523±0.006

3.37±0.04

0.35±0.03 0.20±0.01

--------------------------------------------------------------------------------------------------------------------Lysozyme

6685

0.62

0.481±0.006

3.34±0.04

0.28±0.01 0.24±0.01

--------------------------------------------------------------------------------------------------------------------Ovalbumin

16938 0.69

0.709±0.008

3.10±0.02

0.25±0.02 0.19±0.01

---------------------------------------------------------------------------------------------------------------------

It is instructive to compare the properties presented in Table 3-3, which were determined for proteins, with those that can be additively calculated based on the data on small molecules modeling proteins (20). Our previously determined equilibrium constants, k, for the association of GB with various amino acid side chains in N-acetyl amino acid

117

amides and the glycyl unit (-CH2CONH-) in oligoglycines range between 0.02 M-1 (for the alanine side chain) and 0.32 M-1 (for the phenylalanine side chain) with no pronounced correlation with the type of the constituent atomic groups (20). One can expect that, in the absence of any significant cooperativity, the effective equilibrium constant for the GB-protein interactions should be on the order of 0.17±0.09 M-1, the average value of k evaluated for small protein analogs (20). It should be noted that the affinity of GB for individual atomic groups does not generally follow their polar versus nonpolar pattern but is rather consistent with the scheme reflecting interactions between donors and acceptors of hydrogen bonding, cation--electrons attraction, and other short range interactions (11;23). The most unfavourable interactions are those between GB, lacking hydrogen bond donors, and amide and carboxylate oxygens, while the most favourable interactions are formed between GB and aromatic carbons and amide and cationic nitrogens (23). There is a large difference between the volumetric changes (V0 and KS0) accompanying the GB binding to polar and nonpolar atomic groups in low molecular weight model compounds (20). The values of both V0 and KS0 are smaller for nonpolar side chains relative to their polar counterparts (20). In fact, the values of V0 are negative for nonpolar groups and positive for polar ones (20). The average values of V0 are equal to -0.72±0.25 and 1.13±0.53 cm3mol-1 for nonpolar and polar (and charged) moieties, respectively, while the average values of KS0 for nonpolar and polar (and charged) groups are equal to (1.82±0.71)10-4 and (4.37±1.80)10-4 cm3mol-1bar-1, respectively (20). The estimates of V0 and KS0 for a protein with no cooperativity in its interactions with GB can be made based on V0 = nVnp + (1 - n)Vp and KS0 = nKSnp + (1 n)KSp, respectively, where Vnp and Vp are the average V0 contributions of nonpolar

118

and polar groups, respectively; KSnp and KSp are the average KS0 contributions of nonpolar and polar groups, respectively; and n is the nonpolar fraction of the solvent accessible surface area of a protein. Table 3-4 presents the small molecule-based estimates of V0 (second column) and KS0 (third column) for the four proteins studied in this work.

Table 3-4. Changes in Volume, V0 (cm3 mol-1), and Adiabatic Compressibility, KS0 (10-4 cm3 mol-1 bar-1), for GB-Protein Association in an Ideal Solution Estimated on the Basis of Low-Molecular Weight Model Compound Data ------------------------------------------------------------------Proteins

V0 (est.)

KS0 (est.)

------------------------------------------------------------------Cytochrome c

0.128

3.01

------------------------------------------------------------------Ribonuclease A

0.228

2.95

------------------------------------------------------------------Lysozyme

0.282

3.06

------------------------------------------------------------------Ovalbumin

0.231

3.17

Inspection of data in Tables 3-3 and 3-4 reveals a number of important observations. First, the experimentally evaluated association constants, k, range compactly between 0.19 and 0.24 M-1 and are in good agreement with the estimate of 0.17 ± 0.09 M-1 made

119

based on small molecule data. The observed similarity between the measured and predicted association constants suggests that the interactions of GB with native globular proteins do not involve any significant cooperativity. Second, the measured changes in compressibility, KS0, accompanying the binding of GB to proteins, ranging between 2.9710-4 to 3.3710-4 cm3 mol-1 bar-1, agree well with the estimated values of 2.9510-4 to 3.1710-4 cm3 mol-1 bar-1. This observation is also consistent with a limited or no cooperativity in GB-protein interactions. On the other hand, the measured changes in volume, V0, ranging between 0.481 and 0.709 cm3 mol-1, are significantly larger that the predicted values of 0.128 to 0.282 cm3 mol-1. The discrepancy is not unexpected and, most probably, originates from the unaccounted cavity contribution to volume and the fact that the V0 effects of polar and nonpolar atomic groups are opposite in sign. The predicted values of V0, thus, represent a small difference between large numbers and, consequently, are prone to significant errors, being critically sensitive to the values of n used in the calculations. Taken together, our results are consistent with the picture in which the association of GB with native proteins does not involve any noticeable cooperative effects related to the size of the protein and/or the geometry and the chemical nature of the microenvironment of amino acid residues on its surface. The properties of binding of GB to a protein appear to scale with the size of the latter. These results lend support to studies in which the interactions of GB and other cosolvents with proteins have been modeled by small molecules mimicking protein groups (21;22;44-46).

120

Energetic Considerations. The energetics of transfer of a protein from water to a cosolvent solution is linked to the stabilizing and destabilizing effect of protecting and denaturing cosolvents. In general, stabilizing cosolvents are preferentially excluded from proteins in their both folded and unfolded states, while destabilizing cosolvents are preferentially bound to these states (1;3). The preferential exclusion of protecting cosolvents facilitates protein folding because the native state which is characterized by a lower level of exposure of surface groups. In contrast, the preferential binding of denaturing cosolvents shifts the conformational equilibrium towards the unfolded state which has a greater level of surface group exposure. The transfer of a protein from water to a protecting osmolyte is accompanied by a positive change in free energy, while the transfer to a denaturing cosolvent leads to a decrease in free energy. Thus, the sign and magnitude of water-to-cosolvent transfer free energy for a specific protein is a quantitative measure of the protecting or denaturing influence of a particular cosolvent. Our determined equilibrium constants, k, can be used to gain insights into the energetics of interactions GB with proteins. A change in free energy accompanying the transfer of a solute from water to a solvent-cosolvent mixture is given by the sum:

Gtr = GC + GI

(5)

where GC is the differential free energy of cavity formation in a cosolvent solution and water; and GI is the differential free energy of solute-solvent interactions (9;16;18). For a low concentration solute with n/r identical and independent cosolvent binding sites, GI can be calculated from the relationship (20):

121

GI = -(n/r)RT ln(a1r+ ka3)

(6)

We have approximated the experimental data on a1 and a3 presented by Felitsky and Record (10) by an exponential function; a1 = 0.76 + 0.24exp(-a3/24.11) (20). We use this relationship in conjunction with our data on k to calculate the interaction free energies, GI, of the proteins as a function of a3 from Eq. (6). The calculation results are graphically presented in Figure 3-3.

GI, kcal mol

-1

0

-200

-400

-600

-800 0

10

20

30

40

50

60

a3 , M

Figure 3-3. The free energies of direct GB-protein interactions for cytochrome c (black), ribonuclease A (red), lysozyme (green), and ovalbumin (blue) as a function of GB activity.

122

Inspection of Figure 3-3 reveals a very significant decrease in the interaction free energy, GI, upon an increase in GB activity for all the proteins studied here. This observation signifies highly favourable interactions between GB and protein groups. Note that the changes in the interaction free energy, GI, appear to roughly scale with the size of the protein. A favourable change in GI must be compensated by an unfavourable change in free energy of cavity formation, GC, to produce a net positive change in the water-to-GB transfer free energies, Gtr. For example, the free energy of the transfer of lysozyme from water to 2 M GB is on the order ~10 kcal mol-1 as can be estimated from the data on (2/m3)m2 presented by Arakawa and Timasheff (47). The values of GC may be calculated, in principle, based on the concepts of SPT (35;36). Our SPT-based calculations (data not shown) reveal that the values of GC are of the same order of magnitude but opposite in sign to the values of GI. However, as discussed above, such calculations are not of sufficient accuracy to allow reliable evaluation of the water-to-GB transfer free energies, Gtr, for the proteins studied here. One can judge about the balance of forces governing the stabilizing action of GB from comparing the relative values of Gtr, GI, and GC. The positive transfer free energy, Gtr, for lysozyme on the order of ~10 kcal mol-1 originates from the compensation between the interaction, GI, and cavity, GC, terms each on the order of ~200 kcal mol-1 in magnitude. By extension, a similar fine balance of thermodynamic forces governs the water-to-GB transfer free energies of the other proteins. Thus, we conclude that the mode of action of a specific cosolvent depends on the sign of a small difference (~5 %) between two large numbers: the interaction free energy and the free energy of

123

cavity formation. A qualitatively similar inference has been drawn based on the data for low molecular weight model compounds mimicking proteins (20).

3.6 CONCLUSION Water-soluble cosolvents may exert stabilizing or destabilizing influence on proteins thereby modulating their conformational equilibria. However, the balance of thermodynamic forces governing the mode of action of an individual cosolvent is still poorly understood. In the present work, we apply the volumetric measurements to characterizing the interactions of GB with four typical globular proteins differing in size and structural properties (cytochrome c, ribonuclease A, lysozyme, and ovalbumin). Comparison of the parameters of GB association with proteins with similar data obtained on small analogs of proteins suggests an absence of cooperative effects involved in GB-protein interactions. This result lends credence to studies in which protein-cosolvent interactions are modeled on the basis of low molecular weight compounds. We used the equilibrium constants for GB-protein association to calculate the free energy of direct GB-protein interactions, GI. For all the proteins studied here, direct GB-protein interactions are highly favourable and characterized by large negative values of GI. Comparison of the value of GI determined for lysozyme with the estimate of its differential free energy of cavity formation, GC, and the water-to-GB transfer free energy, Gtr, suggests that the stabilizing action of GB is determined by a fine balance (~5 %) between the GI and GC contributions. By extension, we propose that the mode of action of a specific cosolvent is also determined by the compensation between

124

the GI and GC free energy components, two large quantities producing a relatively small difference on the order of 5 % or less.

3.7 SUPPORTING INFORMATION Table 3-S1. The Extinction Coefficients of the Proteins,  (M-1 cm-1), at Various GB Concentrations --------------------------------------------------------------------------------------------------------------------Proteins

, nm

0M

1M

2M

3M

4M

--------------------------------------------------------------------------------------------------------------------Cytochrome c

409

106000

106600

107700

108700

109900

--------------------------------------------------------------------------------------------------------------------Ribonuclease A

278

9460

9550

9610

9660

9770

--------------------------------------------------------------------------------------------------------------------Lysozyme

280

37630

37820

38020

38490

38730

--------------------------------------------------------------------------------------------------------------------Ovalbumin

280

33750

33930

33990

34130

34290

---------------------------------------------------------------------------------------------------------------------

125

Table 3-S2. The Relative Molar Sound Velocities, [U] (cm3 mol-1), of the Proteins at Various GB Concentrations --------------------------------------------------------------------------------------------------------------------[GB], M

Cytochrome c

Ribonuclease A

Lysozyme

Ovalbumin

--------------------------------------------------------------------------------------------------------------------0

2580±20

2680±20

2570±20

7700±60

--------------------------------------------------------------------------------------------------------------------0.5

2510±30

2670±20

2500±20

7540±60

--------------------------------------------------------------------------------------------------------------------1.0

2460±10

2580±60

2400±40

7260±40

--------------------------------------------------------------------------------------------------------------------1.5

2400±20

2510±20

2300±10

6920±50

--------------------------------------------------------------------------------------------------------------------2.0

2310±40

2400±10

2190±60

6500±40

--------------------------------------------------------------------------------------------------------------------2.5

2210±30

2280±80

2070±50

6160±50

--------------------------------------------------------------------------------------------------------------------3.0

2090±10

2200±30

1920±40

5610±40

--------------------------------------------------------------------------------------------------------------------3.5

1990±50

2140±60

1820±30

5200±60

--------------------------------------------------------------------------------------------------------------------4.0

1910±40

2050±50

1710±30

4910±40

---------------------------------------------------------------------------------------------------------------------

126

3.8 FUNDING This work was supported by Grant RGPIN 203816 from NSERC to T.V.C. Y.L.S. acknowledges graduate support from the CIHR Protein Folding Training Program.

3.9 REFERENCES 1. Timasheff, S. N. (1992) Water as ligand: preferential binding and exclusion of denaturants in protein unfolding, Biochemistry 31, 9857-9864. 2. Timasheff, S. N. (1993) The control of protein stability and association by weak interactions with water: how do solvents affect these processes?, Annu. Rev. Biophys. Biomol. Struct. 22, 67-97. 3. Timasheff, S. N. (1998) Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated, Adv. Protein Chem. 51, 355-432. 4. Timasheff, S. N. (2002) Protein hydration, thermodynamic binding, and preferential hydration, Biochemistry 41, 13473-13482. 5. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Living with water stress: evolution of osmolyte systems, Science 217, 1214-1222. 6. Yancey, P. H. (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses, J. Exp. Biol. 208, 2819-2830.

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7. Record, M. T., Jr., Courtenay, E. S., Cayley, D. S., and Guttman, H. J. (1998) Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water, Trends Biochem. Sci. 23, 143-148. 8. Record, M. T., Jr., Courtenay, E. S., Cayley, S., and Guttman, H. J. (1998) Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments, Trends Biochem. Sci. 23, 190-194. 9. Schellman, J. A. (2003) Protein stability in mixed solvents: a balance of contact interaction and excluded volume, Biophys. J. 85, 108-125. 10. Felitsky, D. J. and Record, M. T. (2004) Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface, Biochemistry 43, 9276-9288. 11. Capp, M. W., Pegram, L. M., Saecker, R. M., Kratz, M., Riccardi, D., Wendorff, T., Cannon, J. G., and Record, M. T. (2009) Interactions of the osmolyte glycine betaine with molecular surfaces in water: thermodynamics, structural interpretation, and prediction of m-values, Biochemistry 48, 10372-10379. 12. Auton, M., Bolen, D. W., and Rosgen, J. (2008) Structural thermodynamics of protein preferential solvation: osmolyte solvation of proteins, aminoacids, and peptides, Proteins Struct. Func. Bioinf. 73, 802-813. 13. Bolen, D. W. and Rose, G. D. (2008) Structure and energetics of the hydrogen-bonded backbone in protein folding, Annu. Rev. Biochem. 77, 339-362. 14. Auton, M. and Bolen, D. W. (2007) Application of the transfer model to understand how naturally occuring osmolytes affect protein stability, Osmosens. Osmosignal. 428, 397-418.

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15. Tang, K. E. and Bloomfield, V. A. (2002) Assessing accumulated solvent near a macromolecular solute by preferential interaction coefficients, Biophys. J. 82, 2876-2891. 16. Davis-Searles, P. R., Saunders, A. J., Erie, D. A., Winzor, D. J., and Pielak, G. J. (2001) Interpreting the effects of small uncharged solutes on protein-folding equilibria, Annu. Rev. Biophys. Biomol. Struct. 30, 271-306. 17. Lee, S. Y. and Chalikian, T. V. (2009) Volumetric properties of solvation in binary solvents, J. Phys. Chem. B 113, 2443-2450. 18. Lee, S., Shek, Y. L., and Chalikian, T. V. (2010) Urea interactions with protein groups: a volumetric study, Biopolymers 93, 866-879. 19. Chalikian, T. V. (2011) Volumetric measurements in binary solvents: theory to experiment, Biophys. Chem. 156, 3-12. 20. Shek, Y. L. and Chalikian, T. V. (2011) Volumetric characterization of interactions of glycine betaine with protein groups, J. Phys. Chem. B 115, 11481-11489. 21. Whitney, P. L. and Tanford, C. (1962) Solubility of amino acids in aqueous urea solutions and its implications for the denaturation of proteins by urea, J. Biol. Chem. 237, 1735-1737. 22. Nozaki, Y. and Tanford, C. (1963) The solubility of amino acids and related compounds in aqueous urea solution, J. Biol. Chem. 238, 4074-4081. 23. Guinn, E. J., Pegram, L. M., Capp, M. W., Pollock, M. N., and Record, M. T., Jr. (2011) Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer, Proc. Natl. Acad. Sci. U. S. A 108, 16932-16937.

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CHAPTER 4: Interactions of Urea with Native and Unfolded Proteins: A Volumetric Study

“Reprinted from Journal of Physical Chemistry B, 118, Ikbae Son*, Yuen Lai Shek*, Anna Tikhomirova, Eduardo Hidalgo Baltasar & Tigran V. Chalikian,. Interactions of urea with native and unfolded proteins: a volumetric study, 13554-13563. Copyright (2014), with permission from American Chemical Society”. *I.S. and Y.L.S. contributed equally to this study and, therefore, should be regarded as joint first authors.

Author‘s Contribution: Yuen Lai Shek performed the majority of the circular dichroism spectroscopic, densimetric and ultrasonic velocimetric experiments, participated in data analysis and in the preparation of the manuscript.

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4.1 ABSTRACT We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and -chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and -chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, V0, and compressibility, KT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, V0, and KT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides.

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4.2 INTRODUCTION Chemical denaturation has been a major component of biophysical research for over a century. Consequently, a great deal of effort has gone into understanding the mechanisms of modulation of the equilibrium between the native and unfolded protein species by stabilizing and destabilizing cosolvents.1-5 Although urea is the most common and frequently used cosolvent in protein studies, controversies exist about the molecular nature of its denaturing action. Currently, most researchers are leaning towards the direct as opposed to the indirect mechanism of urea-induced protein denaturation.5-8 The direct mechanism implies the existence of direct van der Waals or hydrogen bonding or other electrostatic interactions between urea and protein groups

9-11

, while, in the indirect

mechanism, urea exerts its influence via perturbation of the structure of water and the subsequent modification of protein-water interactions.12-14 Volumetric properties of solutes provide a wealth of thermodynamic information describing the entire spectrum of solute-solvent interactions.15-26 In particular, in a binary mixture consisting of the principal solvent and cosolvent, the volumetric properties of solutes reflect the differential solute-principal solvent and solute-cosolvent interactions.19,27,28 We have previously developed a statistical thermodynamics-based algorithm that can be used in conjunction with experimental volumetric data to extract the thermodynamic parameters of elementary solute-cosolvent interactions.19,27,28 With this algorithm, we have conducted a systematic characterization of interactions of urea and glycine betaine with low molecular weight model compounds and interactions of glycine betaine with native globular proteins.19,28-30

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Our results collectively suggest extensive interactions of urea with all functional groups of proteins consistent with the direct mechanism of the effect of urea on protein stability.5,9-11,31-33 More recently, our theoretical investigation based on the volumetrically determined parameters for urea- and glycine betaine-protein interactions has revealed that the mode of action of a specific cosolvent is governed by an extremely subtle balance between the thermodynamic contributions of cavity formation and direct solute-cosolvent interactions.34 In this work, we expand this line of research to studying the solute-solvent interactions of four proteins, namely, apocytochrome c, hen egg white lysozyme, ribonuclease A, and -chymotrypsinogen A, in binary water-urea mixtures. Our goal is to understand to what extent the thermodynamic insights we gained from studying low-molecular weight model compounds are applicable to characterizing whole proteins. In particular, this study aims at elucidating if urea interactions with folded or unfolded protein states involves any cooperativity which is absent in low molecular weight compounds mimicking proteins. In this respect, we have previously found that cooperative effects are absent in the interactions of glycine betaine with folded proteins.30 The specific set of proteins was chosen to cover the full range of conformational states accessible to proteins extending from folded to fully unfolded. Apocytochrome c is unfolded, while lysozyme remains fully folded at all concentrations of urea studied here (0 to 8 M). Hence, these proteins do not undergo any urea-induced conformational transitions thereby enabling one to separately study urea interactions with folded (lysozyme) and unfolded (apocytochrome c) protein states. In contrast, ribonuclease A and -chymotrypsinogen A exhibit urea-induced denaturation transitions. When analyzing the volumetric responses of these proteins to an increase in urea concentration,

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we used the thermodynamic parameters determined for urea interactions with folded and unfolded conformational states derived from our studies of lysozyme and apocytochrome c.

4.3 MATERIALS AND METHODS

Materials. The proteins hen egg white lysozyme, holocytochrome c from equine heart, and ribonuclease A and -chymotrypsinogen A both from bovine pancreas as well as urea were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, ON). All the reagents were of the highest purity commercially available. To get rid of salts and/or any other low-molecular weight compounds that may be present in the samples, the proteins were exhaustively dialyzed against distilled water and lyophilized. Apocytochrome c was obtained and purified from the holoprotein following the previously described protocol.35 The heme was removed by acid acetone extraction after the thioether bridges connecting the heme with the polypeptide chain of the protein had been cleaved by silver sulfate in the presence of acetic acid. Following the removal of the unreacted silver sulfate and the cleaved heme by centrifugation, the apoprotein was reacted with 2-mercaptoethanol to get rid of the bound silver. The resulting apoprotein was extensively dialyzed against water at 4 °C. The content of the holocytochrome c was assessed spectrophotometrically by measuring light absorption in the Soret region at 25 °C and using an extinction coefficient, 408, of 106,500 M-1 cm-1.36 Within the limit of the accuracy of our spectrophotometric measurements (