The goal of the proposed work is to use the muscle protein tropomyosin (which in the native state is a parallel, registered, two-chain, Alpha-helical, coiled coil) as a natural model system to answer questions about the sources of protein structural stability and the thermodynamics and kinetics of protein chain folding and assembly. Tropomyosin is, of course, also of great intrinsic interest in Biochemistry, because of its role in the regulation of muscle contraction. Specifically, the proposed work comprises the following. 1) CD data will be collected on the thermal transitions of: the intact protein molecule (for two genetic variant chains, Alpha and Beta); enzymatically or chemically excised segments of the molecule; and the molecule crosslinked at various specific sites. Application is then to be made of the statistical mechanical theory for the helix-coil transition in such coiled coils in order to dissect out the free energy of the inter-helix interaction and its temperature dependence and site specificity. 2) An examination of chain re-folding and re-assembly will be made for tropomyosin in acidic solution, where there are no inter-chain salt links. This will test the hypothesis that such links dictate the parallel assembly of chains and prevent anti-parallel assembly. 3) An experimental examination of the kinetics of the hybridization reaction and of the unfolding reaction will be undertaken. This hybridization occurs at somewhat elevated temperatures in mixtures of genetic-variant coiled coils, i.e. AlphaAlpha + BetaBeta leads to 2AlphaBeta. A newly developed assay for such hybrids will be employed. The unfolding reaction occurs at elevated temperatures and its kinetics will be examined by T-jump techniques. The data will be interpreted in the light of a recent statistical mechanical theory of the process of unfolding. 4) A study of thermal unfolding of tropomyosin and of its excised segments by NMR spectroscopy will be carried out. In this endeavor, advantage will be taken of two-dimensional NMR techniques and of 13-C labeling at sulfhydryl positions in order to probe the transition at particular molecular sites. 5) An attempt will be made to employ affinity chromatography to separate the Alpha- and Beta- variant chains. With current techniques, this separation is a serious bottle-neck in tropomyosin studies.
