Thermodynamic Mechanism of Protein Stabilization: Crowders vs. Osmolytes

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usability. The complex thermal and isothermal transitions of IgGs as well as their irreversibilities pose a significant challenge to the proper determination of parameters describing the stability of IgGs. Here, we have examined the effects of additives on the kinetic stability of IgGs by differential scanning calorimetry (DSC). The thermal denaturation of full-length IgGs was examined by three-step model, simulating the observed three consecutive transitions (one reversible, two irreversible), while the kinetic stability of the corresponding Fab fragments was analyzed according to the Lumry-Eyring model specifying a single irreversible transition. Ten additives were analyzed: polyols (sorbitol, sucrose and trehalose), salts (sodium perchlorate, chloride and sulfate, including arginine) and methylamines (betaine, sarcosine and trimethylamine oxide). Osmolytes (polyols and methylamines) and salts were found to affect the stability of IgGs differently as seen both in different DSC profiles and in the diverse effects of the additives on parameters characterizing the irreversible step. Our findings highlight the influence of both the additives as well as stabilizing mutations in the Fab fragment on the kinetic stability of the whole IgG. These conclusions could only be reached through proper analyses of thermal denaturations of IgGs using the appropriate model simulating their irreversible thermal transitions, and may help in the development of more predictive strategies for formulation development. Acknowledgements The authors thank Dr. Yuguang Zhao (Wellcome Trust Centre for Human Genetics, Oxford University) for his help in IgG expression and Dr. Peter Gimeson (Malvern Instruments, Uppsala, Sweden) for his help in performing DSC experiments. This work was supported by grants from CELIM (316310) funded by 7FP EU Programs REGPOT and PCUBE. 1054-Pos Board B31 Glucose and Temperature Effect on Human Serum Albumin Structure Minoo Shahani. Islamic Azad University, Tehran medical Branch, Tehran, Iran, Islamic Republic of. Human serum albumin (HSA), due to its long life period, high concentration in plasma and its important roles in body homeostasis is attracted researcher activities for many years. Here HSA-glucose interaction in the range of normal and diabetic concentrations of glucose at 37oC and 42oC is investigated via pH metric, UV absorption spectroscopic and fluorescence methods. The findings indicted that high glucose concentration induces conformations change in HSA as like increment of temperature up to 42 C. It can be concluded that glucose in diabetic condition destabilizes HSA as well as fever. 1055-Pos Board B32 Protective Effect of Pyruvate Against Radiation-Induced Damage in Collagenized Tissues Yuri V. Griko1, Xiaoli Yan2. 1 Life Sciences, NASA Ames Research Center, Mountain View, CA, USA, 2 Clearant Inc., Gaithersburg, MD, USA. Exposure to high doses of ionizing radiation produces both acute and late effects on the collagenized tissues and have profound effects on wound healing. Because of the crucial practical importance for new radioprotective agents, our study has been focused on evaluation of the efficacy of nontoxic naturally occurring compounds to protect tissue integrity against highdose gamma radiation. Here, we demonstrate that molecular integrity of collagen may serve as a sensitive biological marker for quantitative evaluation of molecular damage to collagenized tissue and efficacy of radioprotective agents. Increasing doses of gamma radiation (0-50kGy) result in progressive destruction of the native collagen fibrils, which provide a structural framework, strength, and proper milieu for the regenerating tissue. The strategy used in this study involved the thermodynamic specification of all structural changes in collagenized matrix of skin, aortic heart valve, and bone tissue induced by different doses and conditions of g-irradiation. This study describes a simple biophysical approach utilizing the Differential Scanning Calorimetry (DSC) to characterize the structural resistance of the aortic valve matrix exposed to different doses of g-irradiation. It allows us to identify the specific response of each constituent as well as to determine the influence of the different treatments on the characteristic parameters of protein structure. We found that pyruvate, a substance that naturally occurs in the body, provide significant protection (up to 80%) from biochemical and biomechanical damage to the collagenized tissue through the effective targeting of reactive oxygen species. The recently discovered role of pyruvate in the cell antioxidant defense to O2 oxidation, and its essential constituency in the daily human diet, indicate that the administration of pyruvate-based radioprotective formulations may provide safe and effective protection from deleterious effects of ionizing radiation.

1056-Pos Board B33 The Effect of Hofmeister Ions on the Folding Pathway of Cytochrome C during Thermal or Chemical Denaturization Eric S. Peterson, Sean J. Steinke, Collin A. O’Leary, Mikayla J. Freese. Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls, IA, USA. The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ions from the Hofmeister series is examined. Unfolding was initiated both thermally and with chemical denaturants. Hofmeister ions were added singly and in pairs to alter the stability of the native folded state, the unfolded state, and two partially folded intermediates. Protein stability was characterized by either the midpoint of the chemical denaturization curve or by the melting temperature in the thermal studies. UV/VIS absorption spectroscopy and a basis spectra fitting analysis were used to determine the populations of each protein conformation along the folding pathway. These species can be differentiated by their axial heme ligands. Four species exist in solution: the native HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) intermediates, and the 5C (water) unfolded state. The results indicate that the thermal and chemical denaturization pathways are not the same and that both involve significant backbone rearrangement. The relative populations of the conformational states depends on how the protein is denatured. Additionally, it was found that addition of multiple ions changed the protein’s stability in an additive manner. These results are discussed in terms of the hydrophobic effect, partitioning of the ions to the protein surface, and an altered water structure around the protein. 1057-Pos Board B34 Thermodynamic Fingerprints of the Hofmeister Series - Protein Interactions with Ionic Liquids Michael Senske1, Diana Constantinescu Aruxandei2, Martina Havenith1, Hermann Weinga¨rtner1, Christian Herrmann3, Simon Ebbinghaus1. 1 Physical Chemistry II, Ruhr-University Bochum, Bochum, Germany, 2 School of Chemistry, University of St. Andrews, St. Andrews, United Kingdom, 3Physical Chemistry I, Ruhr-University Bochum, Bochum, Germany. Since Hofmeister ranked salts according to their potential to precipitate hen egg white proteins researchers try to rationalize this Hofmeister series by studying salt effects on protein stability, kinetics, and aggregation [1]. Here, we present the impact of various ionic liquids on the thermal stability of RNase A [2]. With the exception of choline dehydrogenphosphate all ionic liquids tested reduced the melting temperature of RNase A. We compare the results to guanidine hydrochloride, a well characterized protein denaturant. Constructing the Hofmeister series based on the change of the melting temperature, guanidine hydrohochloride takes a medium position. We dissect the free energy changes of RNase A induced by all cosolutes into its enthalpic and entropic contributions [3]. Surprisingly, the enthalpic and entropic mechanisms of all destabilizing ionic liquids are vastly different from the mechanism of guanidine hydrochloride. While the latter causes an enthalpic destabilization, all ionic liquids are entropically destabilizing and enthalpically stabilizing. Thereby, the enthalpic stabilization correlates with an increasing hydrophobicity of the cation. The thermodynamic dissection reveals that Hofmeister series are by no means unique - even for properties of the same system: While the Gibbs free energy and entropy follow the same series as constructed based on the changes of the melting temperature, the series for the enthalpy is inverted. References [1] Ion Specific Hofmeister Effects, Faraday Disc., 2013, Vol. 160. [2] D. Constantinescu et al., Angew. Chem. Int. Ed., 2007, 46, 8887-8889. [3] M. Senske et al., J. Am. Chem. Soc., 2014, 136, 9036-9041. 1058-Pos Board B35 Thermodynamic Mechanism of Protein Stabilization: Crowders vs. Osmolytes Liel Sapir, Daniel Harries. The Hebrew University of Jerusalem, Jerusalem, Israel. Solutes preferentially excluded from macromolecules can drive depletion attractions in important biological association processes. The established Asakura-Oosawa theory relates depletion forces to the excluded volume reduction and the ensuing entropy gain upon macromolecular compaction(1, 2). Accordingly, cosolute-induced protein stabilization is often described in terms of entropically driven ‘‘molecular crowding’’(3). In agreement, many experiments of protein folding and other macromolecular processes suggest that depletion forces are predominantly entropic for some cosolutes, such as polyethylene glycol polymers. However, for other cosolutes, such as polyol osmolytes, the effect is enthalpically dominated, while the entropic change can even be unfavorable(4). Using the Kirkwood-Buff theory we demonstrate that depletion forces can be quantified using the effective interaction between cosolute

Monday, February 29, 2016 and macromolecule. (5) Specifically, by incorporating interactions beyond hard-core, the depletion force attains considerable enthalpic contributions. This analytic theory, along with Monte-Carlo simulations, trace the origins of enthalpically dominated depletion forces to ‘‘soft’’ cosolutemacromolecule repulsions(6). Moreover, these depletion forces can be entropically disfavoured if the effective cosolute-macromolecule interaction consistes of an entropic attractive component and an enthalpic repulsive component(5). Finally, a theoretical mean-field model based on the Flory-Huggins solution theory allows to further trace this effective cosolute-macromolecule interaction to the underlying pairwise interactions in solution(7). 1. Asakura, S., and F. Oosawa. 1954. J. Chem. Phys. 22: 1255-1256. 2. Asakura, S., and F. Oosawa. 1958. J. Polym. Sci. 33: 183-192. 3. Minton, A. 1981. Biopolymers. 20: 2093-2120. 4. Sukenik, S., L. Sapir, and D. Harries. 2013. Curr. Opin. Colloid Interface Sci. 18: 495-501. 5. Sapir, L., and D. Harries. 2015. Curr. Opin. Colloid Interface Sci. 20: 3-10. 6. Sapir, L., and D. Harries. 2014. J. Phys. Chem. Lett. 5: 1061-1065. 7. Sapir, L., and D. Harries. 2015. J. Chem. Theory Comput. 11: 3478-3490. 1059-Pos Board B36 High Molecular Mass Crowders Change the Folding Pathway of D-Glucose/D-Galactose-Binding Protein Alexander V. Fonin1, Serge A. Silonov1, Asia K. Sitdikova1, Irina M. Kuznetsova1, Konstantin K. Turoverov1,2. 1 Institute of Cytology of Russian Academy of Science, Saint-Petersburg, Russian Federation, 2St. Petersburg State Polytechnic University, SaintPetersburg, Russian Federation. In vivo proteins exist in molecular crowding conditions, i.e. when free volume is significantly limited. In vitro such conditions are simulated by concentrated solutions of ‘‘inert’’ polymers (crowders or crowding agents). It was shown that such environment can affect proteins folding, structural dynamics and functional activity. Here, the effect of one of crowders, polyethyleneglycol (PEG) on the folding/unfolding of D-glucose/D-galactose-binding protein (GGBP) was studied. It was shown that PEG 12 and 4 kDa at high concentrations promote the increase of GGBP secondary structure content and shift of GGBP denaturation curves to higher concentrations of chemical denaturant guanidine hydrochloride (GdnHCl). The dependences of GGBP molar ellipticity at 222 nm on GdnHCl concentrations have local minimum near 2 M GdnHCl in these PEG solutions. It may indicate the existence of intermediate state of GGBP with higher content of secondary structure in comparison with the unfolded protein in these GdnHCl concentrations. It was established that denaturation and renaturation curves of GGBP in 12 and 4 kDa PEG solutions with concentrations 300 and 200 mg/ml do not coincide. It was shown that in solutions with low GdnHCl concentrations CD spectra of GGBP after renaturation in the presence of PEG 12 and 4 kDa at high concentrations significantly differ from CD spectra obtained after denaturation of protein in same conditions. All obtained data suggest that PEG of high molecular mass (12 and 4 kDa) at high concentrations (300 and 200 mg/ml) promote the change of protein free energy landscape and folding/unfolding pathway of GGBP. This work was supported by a grant from Russian Science Foundation RSCF 14-24-00131 and RF President fellowship SP-1725.2015.4 (A.V.F.). 1060-Pos Board B37 Probing the Thermal Stability of Lysozyme in Crowded Environments: Tracking Lindemann Criterion Marina Katava1, Guillaume Stirnemann1, Simone Capaccioli2, Alessandro Paciaroni3, Fabio Sterpone1. 1 Laboratoire de Biochimie The´orique, Institut de Biologie PhysicoChimique, Paris, France, 2Department of Physics, Univeristy of Pisa, Pisa, Italy, 3Department of Physics, University of Perugia, Perugia, Italy. Our work focuses on determining the effect of crowded environment and different solvents on the thermal stability of the protein Lysozyme [1] placed in a dilute water solution, dehydrated protein powder, and in a protein powder glycerol solution, the latter two representing crowded environments. The ultimate goal of our work is to probe the validity of the Lindemann criterion for protein melting [2, 3]. We employ an enhanced sampling Molecular Dynamics technique, REST2 [4, 5], where mutually exchangeable protein replicas are simulated at different effective temperatures, achieved by rescaling the force-field potential energy terms. The simulations are paralleled with Elastic Incoherent Neutron Scattering experiments. We first estimate the in silico melting temperature of our systems and reconstruct the stability curves. Instructed by this information, we calculate the scaling of atomic fluctuation approaching melting. Our results show that the atomic fluctuations of different Lysozyme systems converge to similar values approaching the melting temperature, which agrees both with the experimental

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results [unpublished] and the Lindemann criterion [2, 3]. Furthermore, we show that the thermal stability of the hydrated Lysozyme is increased in the presence of crowders (powder) and even more so if glycerol is present in addition to the crowders. The molecular factors, excluded volume versus specific interactions, will be discussed as source of the stability shifts. [1] H.-X. Zhou, G. Rivas, A.P. Minton, Annu. Rev. Biophys. (2008) 37, 375-397. [2] C. Chakravarty, P.G. Debenedetti, F.H. Stillinger, J. Chem. Phys. (2007) 126, 204508. [3] Y. Zhou, D. Vitkup, M. Karplus, J. Mol. Biol. (1999) 285, 1371-1375 [4] L. Wang, R.A. Friesner, B.J. Berne, J. Phys. Chem. B (2011) 115, 94319438. [5] G. Stirnemann and F. Sterpone, submitted to J. Chem. Theory Comput. (2015). 1061-Pos Board B38 Crowding and Protein Dimerization Alex J. Guseman, Stephen T. Lanier, Gary J. Pielak. Chemistry, University of North Carolina Chapel Hill, Chapel Hill, NC, USA. In cells, proteins are surround by macromolecules at concentrations of greater than 100 g/L, yet the majority of our knowledge comes from experiments conducted in dilute buffer solutions. The structure and stability of proteins in cells is influenced by two interactions, hard core repulsions, which arise from excluded volume effects, and transient chemical interactions with surrounding molecules. High concentrations of inert polymers, small molecule cosolutes, and proteins are often used to mimic cellular conditions. Here, we describe the effects of crowding on a protein-protein interaction by using 19F NMR spectroscopy and a variant of the 6 kDa globular B1 domain of protein G. The A34F variant was previously shown to form a side by side dimer in buffer. Using the 3-flourotyorsine labeled variant we measured a dissociation constant of 59 5 5 mM, consistent with previous the study. We then proceed to show the influences of co-solutes on dimer dissociation. Crowding agents such as sucrose, Ficoll-70, glycine betaine, trimethylamineoxide, and bovine serum albumin stabilize the dimer, whereas urea, ethylene glycol, 8 kDa polyethylene glycol and lysozyme destabilize the dimer. Measuring the temperature dependence of dimerization allows for us to measure the van’t Hoff enthalpy of dimer formation. We are now extending this methodology to in-cell NMR studies of the dimer. 1062-Pos Board B39 Protein-Protein Interactions and Secondary Structure affect Helix Stability in Crowded Environments Alan van Giessen, Bryanne Macdonald, Pho Bui. Chemistry, Mount Holyoke College, South Hadley, MA, USA. The dense, heterogeneous cellular environment is known to affect protein stability through interactions with other biomacromolecules. The effect of excluded volume due to these biomolecules, also known as crowding agents, on a protein of interest, or test protein, has long been known to increase the stability of a test protein. Recently, it has been recognized that attractive proteincrowder interactions play an important role. These interactions affect protein stability and can destabilize the test protein. Here, we use multicanonical molecular dynamics and a coarse-grained protein model to study the folding thermodynamics of a small helical test protein in the presence of crowding agents that are themselves proteins. In order to separate the roles of crowder hydrophobicity and secondary structure, two series of simulations were conducted. Each series covered a range of crowder hydrophobicities: in one series the crowding agents were alpha-helices; in the second, they were beta-hairpins. Our results show that the stability of the test protein depends on both the hydrophobicity and secondary structure of the surrounding biomolecules. For hydrophilic crowding agents, the peptide is stabilized through entropic factors, for moderately hydrophilic crowders, the peptide is destabilized through crowderinduced stabilization of its unfolded states, and for hydrophobic crowders, the peptide is either stabilized through packing of crowding agents around the peptide or destabilized due to beta-sheet formation. 1063-Pos Board B40 Investigation on Structural Features and Antiaggregation Properties of Chaperonins and Chaperon Like Molecules Maria Rosalia Mangione1, Dario Dpigolon1, Rosa Passantino1, Rita Carrotta1, Fabio Librizzi1, Caterina Ricci1, Maria Grazia Ortore2, Annalisa Vilasi3, Vincenzo Martorana1, Claudia Marino4, Francesco Cappello5, Pier Luigi San Biagio1, Donatella Bulone6, Silvia Vilasi7. 1 IBF CNR, ce, Italy, 2marche universita, ancona, Italy, 3cnr, napoli, Italy, 4 galveston university, galveston, TX, USA, 5universita`, palermo, Italy, 6ibf, Palermo, Italy, 7IBF CNR, palermo, Italy. Molecular chaperones play essential and several roles in many cellular processes, including protein folding, targeting, transport, and are essential in