The investigators objective is to determine the physical and molecular basis for the cooperativity found between the troponin-tropomyosin complexes which make up the regulatory strand of the thin filament. To this end, methods have been developed or are under development to manipulate the regulatory proteins of chemically skinned mammalian muscle fibers. Troponin C, I, T and tropomyosin will be extracted or replaced either singly or in various combinations with structurally different isomers or with chemically modified ones purified from various muscles. When extraction without replacement is possible, the effect of extraction on cooperativity during thin filament activation by Ca2+ (slope of the pCa/tension relationship) and by rigor crossbridges (slope of the pS/tension relationship) will be examined. Through replacement techniques, fluorescent probes or other structural modifications will be introduced into the regulatory strand of skinned fast and slow muscle fibers. Fluorescence changes in single fibers will be used to measure Ca2+ binding, crossbirdge binding and the state of the regulatory strand simultaneously with tension. The apparatus developed for these studies automates solution mixing, protocol execution, and the collection of fluorescence and tension data; similar apparatus without the fluorescence measurement capability is used to develop better methods of exchanging the regulatory proteins of the thin filament. The cooperative regulation of thin filaments will be modeled with the classical formalism for allosteric proteins and with an induced shift formalism under development. By comparing pCa/tension and pS/tension data from control, extracted, and reconstituted fibers with the model expectations, experimental tests of proposed cooperative mechanisms are developed and new insights into the relationship between molecular form and thin filament physiology are realized. The cooperativity found between the regulatory complexes of the thin filament is more extensive than any molecular interactions hitherto described; it integrates the actions of at least 75 regulatory protein molecules and unifies control over hundreds of actin molecules. To explain it in physical terms may require the development of new ways to envisage molecular interactions in biological structures. Through this contribution to basic knowledge, the proposed work will contribute to the capacity of biomedical researchers to develop treatments for disease.
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