This proposal concerns the mechanism controlling the repetitive contraction and relaxation of the heart, at the level of the protein assemblies that comprise the contractile apparatus. Cyclical variation in the calcium concentration causes calcium binding to and dissociation from troponin, which interacts with the other thin filament proteins tropomyosin and actin so that the thin filament switches between states that will or will not support contraction. A comparable system also exists in skeletal muscles. The long term goal of this work is to understand how troponin and tropomyosin exert this direct regulation, because of its fundamental physiological importance, because this regulation is altered in disease states, and because this is a potential target for therapy. Tropomyosin has an extended coiled-coil structure, and in striated muscles it spans seven actins. We will examine striated muscle alpha-tropomyosin segment by segment, to test hypotheses concerning its function and structure, to circumvent previous difficulties in understanding tropomyosin when using the entire molecule, and to evaluate evidence for major functional heterogeneity within this elongated protein. This will involve host-guest studies of function, and additionally both structural and functional studies of tropomyosin fragments. The structure and conformation of the thin filament also will be investigated by electron microscopy with 3-D reconstruction. This will involve filaments with altered forms of tropomyosin or troponin C (the calcium binding subunit), and short actin filaments created with the filament severing protein gelsolin. Solution studies of these altered filaments will be correlated with the structural results, and used to investigate the conformational transitions of the thin filament, and the spatial propagation of these transitions. Further, permeabilized muscle fibers containing altered forms of TnC will be used to investigate the mechanism of calcium-dependent cooperativity in the intact sarcomere. Finally, the mechanism of cooperative thin filament activation also will be investigated with statistical mechanical modeling of the effects of the non-homogeneous relationship between the regulatory proteins and the seven actins with each regulatory unit.
