Early Events in Protein Folding

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DESCRIPTION (provided by applicant): The central objective of this proposal is to characterize the dynamics and molecular mechanisms of the early events in protein folding. We seek to determine the characteristic rates and underlying molecular mechanisms of the fundamental processes, including secondary structure formation, hydrophobic collapse and formation of specific tertiary interactions. The basic questions we plan to address include: What do the dynamics tell us about the energy landscape of folding? What is the role of local versus nonlocal interactions in shaping the energy landscapes and thus the folding dynamics? How do the early events influence subsequent events in the folding process? Beyond the fundamental importance of the dynamics to the understanding of protein folding mechanisms, is there intrinsic biological significance of rapid protein folding? Fundamentally new approaches to the rapid initiation and structure specific characterization of folding reactions are required to fulfill this objective. We have continued to pioneer the development of such approaches in the last grant period, establishing the viability of laser-induced temperature jump and pH jump coupled with time-resolved spectroscopic methods. We emphasize well-established time-resolved infrared (TRIR) and fluorescence techniques as structure-specific probes of folding dynamics. We propose to address the issues outlined above in a hierarchy of systems: small, de novo model peptides (helix, beta-sheet, turns, coiled-coils) to determine the dynamics of the primary processes; small helical proteins including the engrailed homeodomain (En_HD), the B domain of protein A (BdpA) and apomyoglobin (apoMb); and a fast folding b-sheet protein, the major cold shock protein of E. coil (CspA). The model peptide studies provide an essential foundation for the interpretation of the early events in the protein studies. Implicit in these efforts is the continued improvement of the fast initiation methods and time resolved structurally specific spectroscopic techniques. We expect the results to illuminate both the specific details of folding in these systems, as well as more general principles of folding. The following specific aims will be pursued: l) Determine the dynamics of secondary structure formation in peptide models; 2) Investigate the folding dynamics of small, single domain helical proteins. 3) Investigate the folding dynamics of a small beta-sheet protein, CspA.