Dynamics-stability relationship in cytochromes c

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DESCRIPTION (provided by applicant): Research on highly stable proteins from organisms that thrive at high temperatures (thermophiles) is integral to elucidating the factors that control protein stability and folding. Despite over two decades of research on proteins from thermophiles, the evolutionary strategies behind high-temperature adaptation remain enigmatic. One hypothesis that has emerged is that proteins from thermophiles display enhanced conformational rigidity at room temperature (in which they are in a "cryobiological" state) in comparison with homologous proteins from mesophiles (organisms adapted to 20 - 40 C). In contrast, recent computational studies suggest that some highly thermostable proteins have enhanced flexibility on short time scales (ps - ns). This short time scale flexibility is proposed to enhance protein stability by increasing the entropy of the folded state. Despite the high level of interest in the relationship between protein stability and dynamics, the dynamics of a limited number of proteins from thermophiles have been studied to date. In particular, there are few investigations of short time scale motions (less than micro s). Currently, no NMR characterization of the dynamics of a structurally homologous thermophile-mesophile pair through a wide range of time scales (ps - ms and longer) has been reported. Clearly, more data are needed to understand how flexibility and thermostability are related. The long-range goal of this research is to gain fundamental insight into the relationship between protein dynamics and stability. Our guiding hypothesis is that nature uses the following strategies for stabilizing proteins from thermophiles: 1) Suppression of local. subglobal and global conformational fluctuations throughout the protein on long (greater than us) time scales. 2) Allowing greater local conformational dynamics on short (less than about us) time scales. We will test this hypothesis by characterizing the dynamics of two structurally homologous cytochromes c with differing stabilities (one from a thermophile, and one from a mesophile) and mutants with altered stabilities. Hydrogen exchange studies will be employed to characterize long time scale dynamics (us -ms), and 15N relaxation studies will reveal short time scale dynamics (ps - ns, and longer). Hydrogen exchange data also will be used to determine subglobal stabilities of structural units of these cytochromes. Success in this project will provide fundamental information on the relationship between stability and dynamics of two proteins from a well-studied class and bring us closer to the goal of designing highly stable protein mutants.