Relationships of Protein Dynamics to Structure and Stability

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The objective of this project is to understand the relationship of dynamics to structural features and fold stability in a small model protein, the B1 domain of Streptococcal protein G. This will be accomplished by using NMR relaxation data to compare the backbone NH and side chain methyl group dynamics in various mutants of the B1 domain. First, for a series of 10 single-site mutants, the dynamics at the mutation site and remote sites will be correlated with the properties (volume, branching, and polarity) of the amino acid at the mutation site. Second, the backbone and side chain conformational entropy of each of the 10 mutants will be estimated from the dynamics data and correlated with the fold stabilities of the mutants. This section will incorporate dynamics studies of the folded as well as unfolded forms of the mutants. Third, the extent of correlated motions in the B1 domain will be investigated by analysis of the covariation of internal dynamics parameters. Correlation data will be obtained both for the family of 10 mutants and for the dynamics of the B1 domain measured at several temperatures. Fourth, the mechanism by which dynamic information is propagated from a mutation site to a remote site in the B1 domain will be investigated using double-mutant experiments. Finally, the influence of secondary structural environment on amino acid dynamics will be investigated using two B1 domain variants, in which the same 11-amino acid sequence exists in alpha-helical or beta-hairpin structures, respectively.The chemical properties of proteins such as structural stability, binding to other molecules, and ability to catalyze reactions can often not be adequately understood from the static protein structure because these properties are influenced by the dynamics of the protein. The broad goal of this study is to understand the relationship of dynamics to structural features and fold stability in proteins. The study will provide insights that will aid in our understanding of protein stability and function and may be useful in the design of proteins with novel properties. The major educational goal of this project is the training of a graduate student and a postdoctoral research associate. Activities in the project range from site-directed mutagenesis and protein expression and purification to NMR spectroscopy, other physical biochemistry experiments and extensive computational data analysis. The project will involve development of skills and knowledge in all of these practical areas as well as in theoretical aspects of protein stability and in experimental design.