D-Galactose-Binding Protein

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

213a

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