Collections > Electronic Theses and Dissertations > A Nuclear Magnetic Resonance Investigation of the Role of Structure and Dynamics in the Function of Chymotrypsin Inhibitor 2
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The concept of proteins as static folded three-dimensional objects has yielded to the more sophisticated idea that proteins are better understood as dynamic entities constantly fluctuating about an average conformation rather than being restricted to any single conformation. With the knowledge that proteins sample many different conformational substates comes the question of whether the intrinsic conformational dynamics plays a role in protein function. The work presented in this dissertation begins to explore this question through the study of two model systems. The effects of subtle hydrophobic core mutations on the structure and internal dynamics of the nonallosteric protein chymotrypsin inhibitor 2 (CI2) were characterized using advanced nuclear magnetic resonance methodologies. These experiments revealed that the dynamics of methyl-bearing side chains respond globally and uniformly to hydrophobic core mutations without apparent regard for the position or chemical nature of the mutation; this is a clear example of long range intraprotein communication, a general property that forms the basis of the more complex phenomenon of allostery. Furthermore, the global increase in side-chain flexibility upon mutation takes place without concomitant structural perturbations. Functional assays of these mutants, however, revealed only subtle changes in inhibitory ability, and thus it appears that CI2 is not evolutionarily optimized to harness internal dynamics to modulate function despite its ability to sense perturbations through altered dynamics. The second model system studied is chemotaxis protein Y (CheY), the response regulator domain of the bacterial chemotaxis pathway. CheY is an allosteric protein, and this work reports a characterization of the dynamics involved in phosphorylation-based allosteric activation. The initial results indicate that the classic framework for understanding CheY allostery is too rigid in its definition of distinct inactive and active conformational states. Many of the key residues used to delineate the allosteric state of CheY are dynamic on multiple time scales, thus indicating that they sample numerous conformations and precluding the definition of a sole inactive or active conformation. The results of this work will be useful as a framework for analysis of larger, more complex proteins of biomedical relevance as our theoretical understanding of and experimental capabilities for studying protein dynamics continue to increase.