Structural and Kinetic Characterization of Barriers and Intermediates in Folding of Cytochrome c

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RoderMCB 0079148The goal of this project is to provide a thorough understanding of the structural and energetic factors that govern the spontaneous folding of globular proteins. The kinetic mechanism of folding and unfolding of a small single-domain protein, horse cytochrome c, will be investigated by combining site-directed mutagenesis with ultra-fast mixing techniques, hydrogen exchange labeling and NMR. In order to identify critical interactions involved in stabilizing early intermediates and the transition state ensembles during folding, hydrophobic residues throughout the a-helical core will be altered, and the resulting changes in equilibrium and kinetic parameters will be measured as a function of denaturant concentration. A second series of cytochrome c variants will be prepared to test the hypothesis that polar tertiary interactions are formed late in folding and contribute to the cooperativity of the native structure. Quantitative analysis of the kinetic results for each mutant will provide information on the involvement of individual residues at various stages of folding, including the initial collapse of the chain on the microsecond time scale, the subsequent acquisition of partially structured states and the rate-limiting formation of the native structure. Complementary information on H-bonded structure in early folding intermediates will be obtained by hydrogen exchange labeling and NMR methods. Protection of individual amide protons against H/D exchange on the submillisecond time scale will be measured by combining a sensitive burst-phase labeling protocol with a novel capillary mixing device.The structural insight into intermediates and transition state ensembles thus obtained will identify key interactions involved in their stabilization, which is an essential step toward understanding the sequence determinants for folding and stability of this protein. The findings will elucidate fundamental issues in protein folding, including a) the properties and origin of the kinetic barrier encountered during the initial collapse of the polypeptide chain, b) the role of intermediates in directing folding, c) the nature of the rate-limiting energy barrier and d) the cooperativity in folding/unfolding transitions. The mutational analysis will provide a critical test for the hypothesis that conserved hydrophobic contacts in the core of globular proteins are formed early in folding and are important for efficient folding, while specific side chain packing and polar tertiary interactions are established late in folding and are important for the rigidity and cooperativity of the native structure. The results will provide a firm experimental basis for testing theoretical and computational models of protein folding, fold recognition, structure prediction and de novo protein design.