The mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to the functional, native conformation is not known in detail for any protein. The goal of the proposed research is to investigate both thermodynamic and kinetic aspeCts of the folding mechanism of the alpha subunit of tryptophan synthase, a member of the alpha/beta barrel class of proteins. A unique feature of the alpha subunit is the existence of two stable, partially-folded forms which appear at high concentrations during the urea-induced unfolding reaction. Absorbance, circular dichroism, fluorescence, and NMR spectroscopy will be used to probe general and specific structural features of these folding intermediates. Ultracentrifugation and x-ray scattering will be used to monitor the state of association of these intermediates and provide an estimate of the their radii. Mutational analysis will highlight residues which are involved in stabilizing the various folded forms and to test for interactions between pairs of side chains. Thermodynamic analysis will provide measurements of the relative enthalpies, entropies, and heat capacities of the native, intermediate and unfolded forms. Kinetic experiments will be used to determine these thermodynamic parameters for the transition states which link these stable forms and for transient intermediates which also appear during folding. Additional kinetic experiments will follow the formation of secondary and tertiary structure and of nonpolar surfaces by stopped-flow optical spectroscopy, including time-resolved fluorescence spectroscopy. Fragments of the alpha subunit will be produced by molecular biology or chemical cleavage methods and their structures, stabilities, and kinetic folding properties investigated. Comparisons of the behavior of these fragments with that of the full length protein offers the possibility of simplifying the folding reactions and permitting the assignment of specific kinetic phases to particular regions of the polypeptide. The stable, partially-folded forms of the alpha subunit offer an unprecedented opportunity to study the relationship between and the development of their structural and thermodynamic properties during folding. The insight obtained on the protein folding problem from the proposed studies should impact basic biochemistry, the Human Genome Project, the production of polypeptides in the biotechnology industry, and the ab initio design of proteins with tailored functions.
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