Molecular insights into the role of arginine on protein stabilization

MOLECULAR INSIGHTS INTO THE ROLE OF ARGININE ON PROTEIN STABILIZATION DHAWAL SHAH (B. Tech., IIT-Roorkee) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMICAL AND PHARMACEUTICAL ENGINEERING (CPE) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS This work would not have been possible without the help, guidance, and encouragement by several people. I would like to take this opportunity to express my gratitude towards them. My main thesis supervisor, Prof Raj Rajagopalan, has immensely helped me, both personally and professionally, throughout my research work. His critical thinking, excellent writing skills, timely comments, and in-depth reasoning have greatly improved the quality of this work. I would also like to jointly thank Prof Raj and Prof Trout for providing me a research area to work on. I am also grateful to Prof Saif Khan for his time, discussions, and regular encouragements. I am very grateful to Dr. Pagalthivarthi from IIT. who has not only inspired me to go for a Ph.D., but has also guided me selecting proper university and project. His teachings have greatly improved my character. If you find any good in me, it is all because of following his teachings. Without his guidance and support, this work would not have been possible. I am also indebted Prof. A. Mittal, who taught me how to critically read a research paper, and to Dr. Roy for his regular encouragements and inspirations. Finally, I must thank Singapore-MIT Alliance and National University of Singapore for the financial and the administrative support; Dr. Li Jianguo and Dr. Abdul Rajjak Shaikh for assisting me in the research work; Vignesh, Shankari, Karthik HS, Manju, Soren, Nicholas, Kat, Reno, Srivatsan for creating a wonderful research environment; my friends Vipin, Ruchir, Arpan, Karthik, ii Shrikant, Amit, Sumeet and others for their moral support; and my parents for their encouragements. iii TABLE OF CONTENTS Acknowledgements i Summary viii List of tables x List of figures xi List of symbols xvi 1. Introduction 1.1. Therapeutic proteins and aggregation 1.2. Additives for protein stabilization 1.3. On the use of arginine 1.4. Motivation for the present study and objectives 1.5. Structure of the thesis 13 2. Literature review 2.1. Outline of the chapter 14 2.2. Protein aggregation: classification & pathways 14 2.3. Factors affecting protein aggregation 16 2.4. Arginine stabilizes proteins against aggregation 20 2.5. Suggested mechanisms for arginine-induced protein 23 stabilization 2.5.1. Preferential interactions 23 2.5.2. Surface tension 30 2.5.3. Solubility of amino acids 31 2.5.4. Excluded-volume effect and osmotic stress effect 33 iv 2.5.5. Interactions of arginine with proteins 2.6. Summary 3. Effects of additives perspective 38 39 on aggregation: A thermodynamic 3.1. Introduction 40 3.2. Focus of the chapter 40 3.3. Molecular thermodynamic formulation 41 3.4. Details of the specific reaction model 45 3.5. Results and discussion 47 3.5.1. Effect of additives: Interplay between entropy and 47 internal energy 3.5.2. A note on the use of mixture of additives 3.6. Concluding remarks and implications 53 55 4. Effects of arginine on protein aggregation and the role of the guanidinium group 4.1. Introduction 59 4.2. Focus of the chapter 59 4.3. Materials and methodology 61 4.3.1. Material 61 4.3.2. Extent of aggregation 62 4.3.3. Computational methods 63 4.4. Results 4.4.1. Arginine enhances BSA aggregation 65 65 4.4.2. Protein concentration determines enhancement or 67 suppression of aggregation v 4.4.3. Arginine enhances aggregation of BSA and BLG, 70 but not of LYZ 4.4.4. Guanidine also enhances aggregation of BSA and 72 BLG, but has no effect on LYZ 4.5. Discussion 73 4.5.1. Arginine’s interactions with acidic amino acids 75 4.5.2. Role of arginine-acidic amino acid interaction in 77 aggregation enhancement 4.5.3. Arginine’s interactions with aromatic amino acids 4.5.4. Role of arginine-aromatic stabilization amino acid 79 in 80 4.6. Conclusion 82 5. Arginine-aromatic interactions and their effects on arginineinduced protein stabilization 5.1. Introduction 83 5.2. Focus of the chapter 84 5.3. Materials and methods 85 5.3.1. Materials 85 5.3.2. FFYTP solubility 85 5.3.3. Electron spray ionization-mass spectroscopy (ESI- 86 MS) 5.3.4. FFYTP simulations 86 5.3.5. Protein simulations 88 5.4. Results and discussion 89 5.4.1. Arginine binds to the peptide to enhance the 89 solubility (or stability) of the peptide 5.4.2. MD simulations reveal the preference of arginine to 92 the aromatic residues on the peptide vi 5.4.3. Arginine interacts preferentially with the acidic and 99 the aromatic residues on the proteins 5.4.4. The possible role of arginine-aromatic interactions 103 in solubilizing proteins 5.5. Concluding remarks 106 6. Conclusions and future work 6.1. Conclusions 108 6.1.1. Coarse-grained molecular thermodynamic model of aggregation in presence of additives 108 6.1.2. Heat-induced aggregation 110 6.1.3. Arginine’s interactions with aromatic moieties 112 6.2. Recommendation for future work 114 6.2.1. Further insights into the functioning of arginine 114 6.2.2. Design and use of suitable additives 117 References 121 Appendix A A1 Appendix B B1 Appendix C C1 Appendix D D1 Appendix E E1 Appendix F F1 Appendix G: Publications G1 vii SUMMARY Arginine is commonly used as an additive to enhance refolding yield of proteins and to suppress aggregation of proteins. However, the mechanisms through which arginine does so remain largely unexplored. Most of the studies available to-date on arginine-induced stability of protein solutions have focused on the preferential interactions of arginine with the proteins, but such an approach, while highly useful, is not necessarily sufficient to shed light on the specific molecular interactions that arginine has with protein residues. The focus of this thesis is to initiate a mechanistic study of arginine’s role in stabilizing protein against aggregation. Firstly, we have developed a coarse-grained molecular thermodynamic model to extract some guidelines on the effects of an additive on aggregation reaction equilibria. The results show that the entropic effects (i.e., the excluded-volume effect) and the enthalpic effects (preferential attraction or exclusion) could dramatically alter the effects, even qualitatively, depending on the changes in the co-volume and the accessible surface area of the aggregates relative to that of the reacting monomers, thereby highlighting the fact that overall preferential interactions are not clear enough indicators of the effects of additives on aggregation. Next, we present experiments to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. The results show that the enhancement in aggregation is caused only for BSA and β-lactoglobulin, but not viii for lysozyme, indicating that arginine’s preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine’s role as an enhancer or suppressor of aggregation of proteins. Finally, we present experiments and molecular dynamics simulations on the interaction of aromatic residues of proteins with arginine. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. The results show arginine’s preference for both acidic and aromatic residues, in that order. In the case of aromatic residues, we note that cation-π, hydrophobic, and van der Waals interactions promote the alignment of the planar guanidinium group of arginine with the plane of the aromatic ring of the residues. Such an alignment would cause the polar end of arginine to protrude into the solution and to aid in solvating the arginine-aromatic pair, thereby assisting solubilization of aromatic moieties and aiding suppression of aggregation in the case of proteins. Taken together, the work presented here provides new insights into some of the molecular mechanisms behind the effect of arginine on protein aggregation. Further, the approach we describe herein can be extended to provide a method for selecting suitable additives to stabilize a protein based on an analysis of the amino acid content of the protein. ix LIST OF TABLES Table 1.1: Categorizing commonly used additives for therapeutic protein stabilization. Table 5.1: Arginine associates with the aromatic residues predominantly through the guanidinium group. The table shows average number of central atom of guanidinium group (CZ), Aliphatic group (CG), Amine terminal (N), and Carboxylate terminal (CC) of arginine within 0.35 nm of each residue of the FFYTP peptide. Table 5.2: Average number of heavy atoms of arginine within 0.35 nm of the surface residues of lysozyme and insulin. Results based on molecular dynamics simulations of the proteins in the presence of M arginine indicate that arginine interacts preferentially with the aromatic residues over others. Table 5.3: Arginine associates with the acidic and aromatic residues on insulin predominantly through the guanidinium group. The table shows average number of central atom of guanidinium group (CZ), Aliphatic group (CG), Amine terminal (N), and Carboxylate terminal (CC) of arginine within 0.35 nm of each residue of insulin and lysozyme. Percentage surface residues are shown in the brackets. Table A1: Binding energy (Eb at K) and Gibbs free energy of binding (Gb at 298.15 K) of each amino acid with arginine calculated at GGAPW91 level. All the energies are in kcal/mol. Table E1: Heavy atoms of arginine within 0.35 nm of the surface residues of insulin and lysozyme. Data based on molecular dynamics simulations of the proteins in the presence of M arginine. x Rudolph, R. and Fischer, S. (1990). Process for obtaining renatured proteins. United States, Boehringer Mannheim GmbH (Mannheim, DE). Rzepecki, P., Nagel-Steger, L., Feuerstein, S., Linne, U., Molt, O., Zadmard, R., Aschermann, K., Wehner, M., Schrader, T. and Riesner, D. (2004). "Prevention of Alzheimer's Disease-associated Aβ Aggregation by Rationally Designed Nonpeptidic β-Sheet Ligands." Journal of biological chemistry 279(46): 47497-47505. Saunders, A. J., Davis-Searles, P. R., Allen, D. L., Pielak, G. J. and Erie, D. A. (2000). "Osmolyte-induced changes in protein conformational equilibria." Biopolymers 53(4): 293-307. Sawaya, M. R., Sambashivan, S., Nelson, R., Ivanova, M. I., Sievers, S. A., Apostol, M. I., Thompson, M. J., Balbirnie, M., Wiltzius, J. J. W., McFarlane, H. T., Madsen, A. O., Riekel, C. and Eisenberg, D. (2007). "Atomic structures of amyloid cross-β spines reveal varied steric zippers." Nature 447(7143): 453-457. Schellman, J. A. (1978). "Solvent Denaturation." Biopolymers 17: 1305-1322. Schneider, C. P. and Trout, B. L. (2009). "Investigation of Cosolute-Protein Preferential Interaction Coefficients: New Insight into the Mechanism by Which Arginine Inhibits Aggregation." Journal of Physical Chemistry B 113(7): 2050-2058. Schurr, J. M., Rangel, D. P. and Aragon, S. R. (2005). "A contribution to the Theory of Preferential interaction Coefficients." Biophysical Journal 89: 2258-2276. Shah, D., Lee, T. A., Ramakrishnan, V., Jiang, J. and Rajagopalan, R. (2011a). "Effects of polydisperse crowders on aggregation reactions: A molecular thermodynamic analysis." Journal of Chemical Physics 134(6): 064704. Shah, D., Shaikh, A. R., Peng, X. and Rajagopalan, R. (2011b). "Effects of arginine on heat-induced aggregation of concentrated protein solutions." Biotechnology Progress 27(2): 513-520. Shiraki, K., Kudou, M., Fujiwara, S., Imanaka, T. and Takagi, M. (2002). "Biophysical Effect of Amino Acids on the Prevention of Protein Aggregation." Journal of Biochemistry 132(4): 591-595. Shire, S. J., Shahrokh, Z. and Liu, J. (2004). "Challenges in the Development of High Protein Concentration Formulations." Journal of Pharmaceuticals Sciences 93: 1390-1402. 134 Shukla, D., Shinde, C. and Trout, B. L. (2009). "Molecular Computations of Preferential Interaction Coefficients of Proteins." Journal of Physical Chemistry B 113(37): 12546-12554. Shukla, D. and Trout, B. L. (2010). "Interaction of Arginine with Proteins and the Mechanism by Which It Inhibits Aggregation." The Journal of Physical Chemistry B 114(42): 13426-13438. Shulgin, I. L. and Ruckenstein, E. (2006). "Comment to the Editor - A Protein Molecule in a Mixed Solvent: The Preferential Binding Parameter via Kirkwood-Buff Theory." Biophysical Journal 90: 704-707. Sluzky, V., Klibanov, A. M. and Langer, R. (1992). "Mechanism of insulin aggregation and stabilization in agitated aqueous solutions." Biotechnology and Bioengineering 40(8): 895-903. Soto, C. and Castano, E. M. (1996). "The conformation of Alzheimer's β-peptide determines the rate of amyloid formation and its resistance to proteolysis." Biochemical Journal 314(2): 701-707. Soto, C., Castano, E. M., Frangione, B. and Inestrosa, N. C. (1995). "The αhelical to β-strand transition in the amino-terminal fragment of the amyloid β-peptide modulates amyloid formation." Journal of biological chemistry 270(7): 3063-3067. Soto, C., Kindy, M. S., Baumann, M. and Frangione, B. (1996). "Inhibition of Alzheimer's amyloidosis by peptides that prevent β-sheet conformation." Biochemical and Biophysical Research Communications 226(3): 672-680. Speed, M. A., King, J. and Wang, D. I. C. (1997). "Polymerization mechanism of polypeptide chain aggregation." Biotechnology and Bioengineering 54(4): 333-343. Stagg, L., Zhang, S.-Q., Cheung, M. S. and Wittung-Stafshede, P. (2007). "Molecular crowding enhances native structure and stability of α/β protein flavodoxin." Proceedings of the National Academy of Sciences 104(48): 18976-18981. Street, T. O., Bolen, D. W. and Rose, G. D. (2006). "A molecular mechanism for osmolyte-induced protein stability." Proceedings of the National Academy of Sciences 103(38): 13997-14002. Stumpe, M. C. and Grubmuller, H. (2007). "Interaction of Urea with Amino Acids: Implications for Urea-Induced Protein Denaturation." Journal of the American Chemical Society 129(51): 16126-16131. Tantipolphan, R., Romeijn, S., Engelsman, J. d., Torosantucci, R., Rasmussen, T. and Jiskoot, W. (2010). "Elution behavior of insulin on high-performance 135 size exclusion chromatography at neutral pH." Journal of Pharmaceutical and Biomedical Analysis 52(2): 195-202. Tateno, M. and Hagiwara, Y. (2009). "Evaluation of stabilization energies in p-p and cation–p interactions involved in biological macromolecules by ab initio calculations." Journal of Physics: Condensed Matter 21(6): 064243. Timasheff, S. N. (1995). Preferential Interactions of Water and Cosolvents with Proteins. Protein-Solvent Interactions. Gregory, R. N. New York Marcel Dekker Inc: 445-482. Timasheff, S. N. (2002). "Protein-Solvent preferential interactions, protein hydration, and modulation of biochemical reaction by solvent components." Proceedings of the National Academy of Sciences 99: 97219726. Timasheff, S. N. and Xie, G. (2003). "Preferential interactions of urea with lysozyme and their linkage to protein denaturation." Biophysical Chemistry 105: 421-448. Tobitani, A. and Ross-Murphy, S. B. (1997). "Heat-Induced Gelation of Globular Proteins. 2. Effect of Environmental Factors on Single-Component and Mixed-protein gels." Macromolecules 30: 4855-4862. Tokuriki, N., Kinjo, M., Negi, S., Hoshino, M., Goto, Y., Urabe, I. and Yomo, T. (2004). "Protein folding by the effects of macromolecular crowding." Protein Science 13(1): 125-133. Torrent, J., Alvarez-Martinez, M. T., Liautard, J.-P. and Lange, R. (2006). "Modulation of prion protein structure by pressure and temperature." Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1764(3): 546-551. Tsumoto, K., Ejima, D., Kita, Y. and Arakawa, T. (2005). "Review: Why is Arginine Effective in Suppressing Aggregation?" Protein and Peptide Letters 12: 613-619. Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D. and Arakawa, T. (2003). "Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine." Biochemical and Biophysical Research Communications 312(4): 1383-1386. Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J. S. and Arakawa, T. (2004). "Role of Arginine in Protein Refolding, Solubilization, and Purification." Biotechnology Progress 20(5): 1301-1308. Vagenende, V., Yap, M. G. S. and Trout, B. L. (2009). "Molecular Anatomy of Preferential Interaction Coefficients by Elucidating Protein Solvation in 136 Mixed Solvents: Methodology and Application for Lysozyme in Aqueous Glycerol." Journal of Physical Chemistry B 113(34): 11743-11753. Vedadi, M., Niesen, F. H., Allali-Hassani, A., Fedorov, O. Y., Finerty, P. J., Wasney, G. A., Yeung, R., Arrowsmith, C., Ball, L. J., Berglund, H., Hui, R., Marsden, B. D., Nordlund, P. r., Sundstrom, M., Weigelt, J. and Edwards, A. M. (2006). "Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination." Proceedings of the National Academy of Sciences 103(43): 15835-15840. Vemuri, S., Yu, C. and Roosdorp, N. (1993). "Formulation and stability of recombinant alpha 1-antitrypsin." Pharmaceutical biotechnology 5: 263286. Vondrasek, J., Mason, P. E., Heyda, J., Collins, K. D. and Jungwirth, P. (2009). "The Molecular Origin of Like-Charge Arginine-Arginine Pairing in Water." Journal of Physical Chemistry B 113(27): 9041-9045. Wang, W. (1999). "Instability, stabilization, and formulation of liquid protein pharmaceuticals." International Journal of Pharmaceutics 185: 129-188. Wang, W. (2005). "Protein aggregation and its inhibition in biopharmaceutics." International Journal of Pharmaceutics 289: 1-30. Wang, W., Nema, S. and Teagarden, D. (2010). "Protein aggregation--Pathways and influencing factors." International Journal of Pharmaceutics 390(2): 89-99. Wang, Y. and Dubin, P. L. (1998). "Observation of Ficoll charge using sizeexclusion chromatography." Journal of Chromatography A 800(2): 181185. Xie, Q., Guo, T., Lu, J. and Zhou, H.-M. (2004). "The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state." The International Journal of Biochemistry & Cell Biology 36(2): 296-306. 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(4566): 1214-1222. Zhang, R., Hu, X., Khant, H., Ludtke, S. J., Chiu, W., Schmid, M. F., Frieden, C. and Lee, J.-M. (2009). "Interprotofilament interactions between Alzheimer's Aβ-42 peptides in amyloid fibrils revealed by cryoEM." Proceedings of the National Academy of Sciences 106(12): 4653-4658. Zhou, B.-R., Liang, Y., Du, F., Zhou, Z. and Chen, J. (2004). "Mixed Macromolecular Crowding Accelerates the Oxidative Refolding of 137 Reduced, Denatured Lysozyme: Implications for protein folding in intracellular environments." Journal of biological chemistry 279(53): 55109-55116. Zhou, H.-X. (2008). "Effect of mixed macromolecular crowding agents on protein folding." Proteins: Structure, Function, and Bioinformatics 72(4): 11091113. Zhou, H.-X. (2009). "Crowding effects of membrane proteins." Journal of Physical Chemistry B 113: 7995-8005. Zhou, H.-X., Rivas, G. and Minton, A. P. (2008). "Macromolecular Crowding and Confinement: Biochemical, Biophysical, and Potential Physiological Consequences." Annual Review of Biophysics 37(1): 375-397. Zimmerman, S. B. and Minton, A. P. (1993). "Macromolecular crowding: biophysical, biochemical, and physiological consequences." Annual Review Of Biophysics And Biomolecular Structure 22: 27-65. 138 Appendix A Table A1: Binding energy (Eb at K) and Gibbs free energy of binding (Gb at 298.15 K) of each amino acid with arginine calculated at GGAPW91 level. All the energies are in kcal/mol.a a Amino acid Eb 0K Gb298.15K Asp –35.52 –20.84 Glu –34.72 –21.03 Asn –23.81 –12.51 Gln –24.39 –13.69 Trp –17.82 –4.17 Tyr –17.50 –4.18 Phe –17.34 –4.50 Pro –18.01 –4.95 Ser –19.83 –10.03 Thr –18.61 –5.28 Arg –30.57 –20.84 Lys –12.54 –1.23 His –23.11 –10.64 Cys –23.42 –10.10 Met –18.67 –5.14 Ala –15.05 –6.13 Val –13.49 –0.69 Leu –14.31 –1.36 Ile –12.92 –0.04 Both the binding energy and the Gibbs free energy reported herein are inclusive of ZPVE correction. A1 Appendix B Figure B1: Molecular dynamics simulations of C20 fullerene grafted with acidic (−COOH) groups at opposite ends in the presence of arginine. The figures show radial distribution functions and coordination number of the fullerene particles. See Li et al. for simulation details. Both radial distribution function and coordination number indicate that in the absence of arginine there is no aggregation of the particles. However, in the presence of arginine, we observe concentration-dependent aggregation of the particles, with maximum aggregation observed in the presence of 250 mM. The results are qualitatively similar to those observed in our heat- induced aggregation of acidic proteins. Visual observations show formation of the proposed “bridges”. B1 Figure B2: Molecular dynamics simulations in the presence of guanidine. Although in the absence of guanidine there is no aggregation, similar to arginine, we observe enhancement in the aggregation of the particles in the presence of low concentrations of guanidine. At same concentration guanidine enhances aggregation of the particles more than arginine. The results are qualitatively similar to those observed in our experiments. At high enough guanidine concentrations, M, which is generally used to solubilize protein aggregates, the results not show any aggregation. B2 Appendix C CLUSTERING OF ARGININE IN BULK SOLUTION It has been claimed in the literature (Das et al.) that the clustering of arginine in the solution causes inhibition of hydrophobic associations because of the creation of a “hydrophobic environment” in the solution. It is also further speculated therein that the cluster formation occurs due to the alignment of the three methylene groups of arginine with those of the other arginine molecules. In another work (see Li et al.) in our lab we have performed MD simulations of arginine solution at low and normal pH. The MD simulations clearly indicate that arginine molecules form clusters at normal pH but not at low pH, as suggested by experiments of Das et al. However, the results also indicate that arginine form “head-to-tail” clusters due to electrostatic interactions rather than through the alignment of the aliphatic chains. The head-to-tail association at normal pH is reasonable in view of the strong electrostatic interactions between the oppositely charged carboxyl groups (COO−) and guanidinium groups stabilized by double hydrogen bonds; indeed, such an association has been identified in many complexes involving biomolecules. Arginine at low pH (ARGp) does not form clusters because of the weak hydrogen bonding ability of COOH group compared to that of the COO− group, which supports the available experimental reports. The absence of arginine clusters at low pH suggests that the complexes involving multiple hydrogen bonds (e.g., structures formed by two hydrogen bonds; see Figure C1) are critical to maintain stable clusters. C1 Figure C1: One of the stable conformations of arginine in solution. The illustration shows the head-to-tail association mediated by the double hydrogen bonds between polar and guanidinium groups. Further, the MD results on aggregation of hydrophobic particles in the presence of arginine also showed that the clustering of arginine in the bulk solution does not significantly influence the solubilization or suppression of aggregation. However, the self-association of arginine through the “head-to-tail” association mentioned earlier does appear to aid solubilization or suppression of hydrophobic association, not by creating a “hydrophobic environment” in the bulk solution, but by forming a ‘cage-like’ structure around the hydrophobic moieties. For further details, one can refer to our published article (Li et al.). C2 Appendix D Figure D1: Detailed mass spectra of FFYTP peptide at different arginine concentrations. Since the low m/z region (< 550) was crowded by non-peptide peaks, we report data only for m/z beyond 600. (a) M arginine (b) 200 mM arginine (c) 400 mM arginine D1 (d) 600 mM arginine e) 800 mM arginine (f) 1000 mM arginine D2 Appendix E Table E1: Heavy atoms of arginine within 0.35 nm of the surface residues of insulin and lysozyme. Data based on molecular dynamics simulations of the proteins in the presence of M arginine. INSULIN Residue Arginine atoms* ASA** Residue G1 1.59 0.47 K1 T8 1.26 0.66 V2 I 10 0.08 0.43 R5 Y 14 5.85 0.77 E7 Q 15 0.10 0.4 K 13 N 18 5.94 0.67 R 14 N 21 9.51 1.00 D 18 F 22 0.94 0.96 N 19 V 23 0.53 0.63 R 21 N 24 1.09 0.68 Y 23 Q 25 1.26 0.55 F 34 H 26 1.14 0.4 N 37 C 28 0.40 0.41 Q 41 S 30 3.42 0.76 T 43 H 31 1.39 0.5 N 44 V 33 1.40 0.41 R 45 E 34 7.56 0.43 N 46 Y 37 6.25 0.52 T 47 L 38 1.30 0.56 D 48 E 42 7.52 1.00 R 61 R 43 3.35 0.65 W 62 G 44 0.28 0.4 N 65 F 46 10.22 0.81 G 67 T 48 2.51 0.68 R 68 K 50 3.81 0.99 P 70 T 51 7.53 1.00 G 71 R 73 * Heavy atoms of arginine within 0.35 L 75 nm of the residue. N 77 **fraction of the total residues area P 79 exposed to the solvent. S 81 LYSOZYME Arginine atoms* 4.79 6.10 0.61 8.44 5.09 3.23 8.24 3.67 1.35 2.22 1.47 1.55 4.39 11.43 14.09 0.94 7.26 4.82 10.44 0.89 9.83 1.88 2.50 0.87 0.83 2.54 7.97 0.93 0.29 1.01 0.30 ASA** 0.71 0.47 0.76 0.90 0.50 0.92 0.63 0.81 0.95 0.47 0.43 0.83 0.57 0.58 0.50 0.70 0.40 1.00 0.69 0.43 0.47 0.63 0.75 1.00 0.80 0.84 0.69 0.54 0.82 0.49 0.82 E1 S 85 S 86 D 87 T 89 K 97 S 100 D 101 G 102 N 103 N 106 A 107 V 109 R 112 N 113 R 114 K 116 G 117 T 118 D 119 Q 121 A 122 R 125 G 126 R 128 L 129 0.14 0.06 0.52 0.52 0.11 2.96 4.79 2.49 0.14 0.18 0.29 1.12 1.35 0.23 0.74 0.42 0.73 0.40 2.18 2.95 0.00 1.03 0.47 5.60 10.92 0.63 0.64 0.44 0.44 0.62 0.55 0.87 0.79 0.43 0.48 0.43 0.66 0.55 0.80 0.68 0.66 1.00 0.60 0.71 0.70 0.49 0.93 0.93 0.97 0.49 E2 Appendix F Figure F1: The figure compares the effects of arginine and homo-arginine on the heat-induced aggregation of BSA. See section 4.3 for methodology. The results indicate that the aliphatic segment of arginine has negligible role in enhancing or suppressing aggregation. The results also indicate that the size of arginine molecule is not a significant factor in understanding the suppression of protein aggregation. F1 Appendix G PUBLICATIONS JOURNAL ARTICLES 1. Dhawal Shah, Abdul Rajjak Shaikh, Xinxia Peng, and Raj Rajagopalan. “Effects of Arginine on Heat-induced Aggregation of Concentrated Protein Solutions” Biotechnology Progress, 2011, DOI: 10.1002/btpr.563. 2. Jianguo Li, Manju Garg, Dhawal Shah, and Raj Rajagopalan. “Solubilization of Aromatic and Hydrophobic Moieties by Arginine in Aqueous Solutions” Journal of Chemical Physics, 2010, 133, 054902. 3. Dhawal Shah, Aik Lee Tan, Ramakrishnan Vigneshwar, Jiang Jianwen, and Raj Rajagopalan. “Effects of Polydisperse Crowders on Aggregation Reactions: A Molecular Thermodynamic Analysis” Journal of Chemical Physics, 2011,134, 064704. 4. Dhawal Shah, Jianguo Li, Shaikh Abdul Rajjak, and Raj Rajagopalan. “Arginine-Aromatic Interactions and Their Effects on Arginine-Induced Solubilization of Aromatic Solutes and Stabilization of Proteins” Submitted. 5. Dhawal Shah and Raj Rajagopalan. “Effects of Crowding on Aggregation and Association Reactions: A Colloid Physics Perspective” In preparation, Invited article: Encyclopedia of Surface of Colloid Science. CONFERENCES 1. Dhawal Shah and Raj Rajagopalan. “Excipients for Protein Stabilization: Identifying the Molecular Mechanisms” SMA Annual Symposium/International Symposium for Nano-Manufacturing – 2007. 2. Dhawal Shah and Raj Rajagopalan. “Effect of mixture of additives on Biochemical reactions” SMA Annual Symposium – 2008. 3. Dhawal Shah, Shaikh Abdul Rajjak, and Raj Rajagopalan. “How arginine, as an excipient, affects the stability of concentrated protein formulations?” Proteins and Peptide Conference, Beijing – March 2010. G1 [...]... varying arginine concentrations, indicating that the results observed are independent of buffer conditions Figure 4.3: Effect of protein concentration on arginine- induced enhancement of aggregation Figure 4.4: Aggregation rate (defined as being proportional to the maximum rate of change of turbidity for a given set of protein concentration and arginine concentration) of BSA depends on protein concentration... residues) on a protein? What are some of the possible implications of such interactions on arginine- induced protein stabilization? As noted above, protein aggregation and arginine- induced suppression of aggregation are an outcome of myriads of intermolecular and intramolecular interactions However, an understanding of some of these interactions sheds light on some aspects of the mechanism of arginine- induced... aggregation?” There are several reports available in the literature on the effect of arginine on protein aggregation Most of these reports discuss the effects in terms of the preferential interactions of arginine with proteins Other reports attribute the role of arginine to change in the surface tension (Arakawa et al 2007a) or to the specific interactions of the guanidinium group (Ghosh et al 2009) or the. .. Depending on the conditions, a protein can form soluble or insoluble aggregates Various factors affect protein aggregation, such as the protein structure and 14 environmental conditions In the present section we present some of these factors and their role in protein aggregation Before explaining these factors, we first make brief notes on the pathways and kinetics of protein aggregation The pathways of protein. .. arginine- induced protein stabilization In chapter 3 we develop a coarse-grained, molecular thermodynamic model of aggregation to show effects of an additive on aggregation Chapter 4 presents experiments on heat-induced aggregation of three proteins in the presence of arginine and guanidine and discusses the role of guanidinium group of arginine on arginine- induced protein stabilization In chapter 5, we study the. .. interactions predict protein stabilization? Apart from preferential interactions, what other factors can affect the thermodynamics of additive-induced protein stabilization? How do these factors contribute to aggregation or stabilization of proteins? To address the above questions, we use a coarse-grained molecular thermodynamic model based on liquid-state physics to extract some guidelines on the effects of. .. based on their observations on the solubility measurements of various amino acids in the presence of 1 M arginine and guanidine In order to understand the role of the guanidinium group (as well as those of the other segments) we consider the following objective:  Compare and contrast the effects of guanidine and arginine on aggregation of some model proteins Our intent here is to address questions such... does the guanidinium group of arginine interact with the various 11 residues on proteins? What are some of the possible implications of these interactions on protein stabilization? To do so, we study heat-induced aggregation of three commonly studied proteins, viz BSA, lysozyme, and β-lactoglobulin, and examine the effects of arginine and guanidine aggregation In addition, we use density-functional-theory... protein aggregation, such an approach fails to shed light on the specific additive -protein interactions that contribute to protein stability In view of this, in the present work, we focus further on specific molecular interactions of arginine with proteins to gain some insights to arginineinduced proteins stabilization Ideally, a molecular- level picture of interactions of an additive with protein will... to the large additives The effect of additives depends on the relative dimensions of the product and the reactant Figure 3.3: Difference in the excluded volume (EVE) and the accessible surface area (ASA) between the reactant and the product of a dimerization reaction varies with the crowder size Figure 3.4: Effect of varying the strength of interaction on the equilibrium reaction in the presence of . MOLECULAR INSIGHTS INTO THE ROLE OF ARGININE ON PROTEIN STABILIZATION DHAWAL SHAH (B. Tech., IIT-Roorkee) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. 79 4.5.4. Role of arginine- aromatic amino acid in stabilization 80 4.6. Conclusion 82 5. Arginine- aromatic interactions and their effects on arginine- induced protein stabilization 5.1 alignment of the planar guanidinium group of arginine with the plane of the aromatic ring of the residues. Such an alignment would cause the polar end of arginine to protrude into the solution and