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FOR THE RECORD

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

¹ Department of Chemistry, Indiana University, Bloomington, Indiana 47405-0001, USA
² Department of Chemistry and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA

(RECEIVED June 10, 2004; FINAL REVISION August 16, 2004; ACCEPTED August 17, 2004)

"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 ¹³CH₂D 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

Abbreviations: ASA, accessible surface area • G_conf, conformational free energy • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • R₁, longitudinal relaxation rate constant • R₁, transverse relaxation rate constant • S², order parameter • S²_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 http://www.proteinscience.org/cgi/doi/10.1110/ps.04926604.

Reprint requests to: Martin J. Stone, Department of Chemistry, Indiana University, Bloomington, IN 47405-0001, USA; e-mail: mastone{at}indiana.edu; fax: (812) 855-8300.

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