Folding Mechanisms of Dimeric Beta-Barrel Proteins

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DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the mechanisms by which a pair of dimeric beta-barrel proteins, apo-human superoxide dismutase and H1V-1 protease, fold and assemble into their native, functional forms. A combination of continuous-flow, stopped-flow and manual-mixing methods, interfaced to optical detection, will provide access to folding events over the micro- to kilo-second time range. The kinetic and equilibrium data will be fit globally to kinetic and thermodynamic models to enable the extraction of microscopic rate constants for individual steps in the reaction. Information on the size, shape and structure of the transient intermediates will be obtained with small angle x-ray scattering, fluorescence anisotropy, and Forster resonance energy transfer techniques. Native-state and quench-flow hydrogen exchange methods, combined with mass spectrometry, will be utilized to monitor the secondary structure of rare, partially-folded states under equilibrium conditions and the development of secondary structure during folding. The structural information, available from both the optical and hydrogen exchange studies, will be compared to the predictions of Go-like and molecular dynamics simulations. Site-directed mutagenesis at selected positions in beta strands, loops, and at the subunit interface will test the roles of individual side chains on the folding, association and stability of these dimeric proteins. The insights obtained into the structure/mechanism relationship will enhance the extraction of generalities from the sequences and/or sub-structures that guide the reaction over the folding free energy surface. The effects of naturally-occurring mutations in aposuperoxide dismutase on the thermodynamic and kinetic properties of folding may provide useful insights into the molecular basis of amyotrophic lateral sclerosis. More generally, the results of this study are expected to provide models and paradigms by which multi-subunit complexes assemble.