The goal of this research is to elucidate the mechanisms of interaction of the proteins that make up the thin filaments of striated muscle: in particular, to determine the physical and molecular basis of cooperative interactions between the troponin-tropomyosin units which extend the length of the thin filaments of striated muscle. Skinned muscle fibers are used as experimental material because these regulatory proteins and their environment can be manipulated. Turnover rates of the thin filament regulatory proteins will be measured to gain insight into the intermolecular forces which hold the regulatory strands together and in place, and to establish conditions for the nondestructive replacement of proteins. The regulatory proteins,troponins (C,I, and T) and tropomyosin, will be exchanged or extracted and replaced with chemically modified forms, genetically different forms, and forms labeled with fluorescent or electon opaque probes. The hypothesis that rigor crossbridges activate thin filaments will be tested by decorating them with irreversibly bound myosin fragments. An apparatus used in solution mixing, protocol execution, and data collection is automated and under computer control. A similar apparatus built into a spectrofluorometer correlates data from fluorescent molecular probes with tension data. The cooperative regulation of thin filaments will be modeled using the classical formalism for allosteric proteins (Monod el al., l965) and an "induced shift" model. By comparing experimental calcium concentration vs tension and substrate vs tension curves from control, extracted, and experimentally reconstituted fibers with the model predictions, new insights into potential mechanisms of thin filament regulation should be obtained. Although the mechanisms that regulate the contraction of striated muscle have been the object of much study, much remains to be learned at the molecular level of how each of the proteins of the contractile apparatus is involved and how these proteins interact in the process of shortening. This research will extend our fundamental knowledge of the function of these important muscle proteins.
