Protein folding is tightly regulated in living organisms, and the fidelity of this process is essential to the proper functioning of most cellular and extracellular events. A number of diseases including Alzheimer's and Parkinson's are associated with malfunction of endogenous proteins, often as a result of their misfolding or misassemby. Thus, the continued study of factors that influence and stabilize proper protein folding remains an important pursuit. To this end, we propose making chemical modifications to protein backbones to illuminate the dominant forces that mediate protein folding. We will investigate the following specific aims: 1. To assess the contributions of backbone hydrogen bonds to the folding of the three-helix bundle B- domain of Staphylococcal protein A (BdpA) using amide-to-ester mutants. Thermodynamic (thermal and chaotropic denaturation) and kinetic (laser T-jump) evaluations of these mutants and comparison to the native protein are expected to validate three hypotheses. First, H-bonds most shielded from solvent will be stronger than those that are more solvent-exposed. Second, backbone H-bonds in helices contribute more to folding than backbone H-bonds in sheets. Third, kinetic assessment of these mutants will characterize a folding mechanism for BdpA that is consistent with that identified by side chain muatagenesis studies. 2. To probe the requirement for backbone H-bonds using amide-to-E-olefin mutants of BdpA. E-Olefin dipeptide isosteres will be synthesized and inserted into BdpA sequences. These mutants will be analyzed analogously to the amide-to-olefin mutants and correlations between the microenvironment of an H-bond and its contribution to folding are expected to parallel those found for amide-to-ester mutants. 3. To evaluate the efficacy of peptidomimetic turn nucleators to accelerate beta-sheet protein folding. Loop 1, which nucleates folding of native Pin WW domain, will be modified by replacing turn residues with putative peptidomimetic turn nucleators to discern whether this modification hastens folding of the protein relative to the wild type protein. It is hypothesized that introduction of rigid synthetic turn nucleators will accelerate protein folding in both Pin WW domain and a larger protein, TNfnS, relative to the wt protein. A better understanding of the forces involved in protein folding and stability is essential to the development and improvement of therapeutics for protein misfolding and misassembly diseases including Alzheimer's and Parkinson's. The research proposed will illuminate forces that dictate protein folding; in turn, this knowledge will lead to the development of new strategies for treatment of such diseases. ? ? ?
