Understanding the fundamental molecular mechanisms by which proteins fold into the precise three-dimensional structures required for biological activity remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is coded within the amino acid sequence; just how that code is translated into folding pathways and highly specific tertiary structure is not yet known. There is an urgent need for detailed information on the structures of folding intermediates at the level of individual amino acid residues, and a need for a deeper understanding of the factors that influence their rates of formation and stability. The overall objective of the proposed research is to address some of these outstanding issues through kinetic and equilibrium studies of intermediates formed on the folding pathway of apomyoglobin. Apomyoglobin is ideally suited for such studies because a stable molten globule intermediate that is formed early on the kinetic folding pathway can also be obtained under equilibrium conditions appropriate for high resolution NMR experiments. A combination of mutagenesis, stopped-flow kinetics measurements, hydrogen exchange pulse labeling, and state-of-the-art heteronuclear NMR experiments will be applied to investigate the kinetic folding pathway of apomyoglobin and characterize the structure of the stable molten globule folding intermediate. Mutations will be introduced at specific sites to probe the molecular interactions that stabilize the kinetic folding intermediates, and to investigate the effect of alterations in the intrinsic secondary structural propensities of the polypeptide on the rate of formation and stability of the molten globule intermediate. Direct heteronuclear NMR methods will be used to obtain critical information on the structure, dynamics, and state of hydration of the equilibrium molten globule intermediate of apomyoglobin at the level of single residues. This research program is expected to provide important new insights into the structure and stabilization of folding intermediates at an unprecedented level of detail, and will add significantly to the understanding of protein folding mechanisms. In addition, the kinetic folding pathway of an evolutionarily distant plant leghemoglobin will be characterized, along with the structure of a partly folded intermediate. These studies are expected to provide a particularly stringent test of the hypothesis that folding pathways are conserved in homologous proteins.
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