Abstract"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the -sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13 CH 2 D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.Keywords: -sheet propensity; B1 domain; conformational entropy; dynamics; GB1; NMR; protein G; protein stability Supplemental material: see www.proteinscience.orgThe relative stabilities of folded and unfolded proteins are determined by the interplay of many enthalpic and entropic factors. In this communication we describe an experimental study of the role of conformational entropy within the folded state. It is widely appreciated that folded proteins have substantially reduced conformational entropy relative to the unfolded state, yet retain some residual flexibility. Although the reduction in entropy upon folding is thermodynamically unfavorable, it is compensated by the formation of enthalpically favorable interactions and by a favorable increase in solvent entropy. The native ensemble represents the optimal trade-off between these various factors. Mutation of a protein (or ligand-binding, changes in pH, ionic strength, temperature, pressure, etc.) could potentially affect any of these thermodynamic factors. Therefore, to understand the influence of mutations on protein stability, it would be advantageous to understand, among other things, the effect of the mutations on conformational entropy. Abbreviations and symbols: ASA, accessible surface area; ⌬G conf , conformational free energy; HSQC, heteronuclear single quantum coherence; NMR, nuclear magnetic resonance; R 1 , longitudinal relaxation rate constant; R 1 , transverse relaxation rate constant; S 2 , order parameter; S 2 axis , methyl axis order parameter; S conf , conformational entropy; e , internal correlation time.Article published online ahead of print. Article and publication date are at
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