The overall goal of this project is to enhance the understanding of the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to the native conformation. The principal target of a variety of biophysical studies will be the alpha subunit of tryptophan synthase (alphaTS) from E. coli. alphaTS is a single domain protein of 268 amino acids whose sequentially-alternating helices and strands are wound into an alpha/beta barrel motif, one of the most common in biology. A combination of circular dichroism, fluorescence, mass spectrometry, and NMR techniques and protein engineering methods will be use to determine the physical properties of both stable and transient intermediates that have previously been observed to appear during the urea-induced unfolding and refolding of alphaTS. Continuous-flow mixing techniques will be used to explore submillisecond-folding reactions that are known to occur for alphaTS. Mutational analysis will monitor the acquisition of sterospecific packing in the beta-barrel core and test the role of proline isomerization in defining parallel folding channels. The information obtained on the structural, dynamic and thermodynamic properties of these partially folded forms will provide important insights into the progressive development of stability and structure in alphaTS. The generality of the conclusions on the mechanism of folding of alphaTS will be explored for a few other members of the alpha/beta barrel class. A solution to the folding problem would have an important impact on biochemistry, medicine and the biotechnology industry.
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