The long term objectives of this work are to define accurately the structural relationships of actin, myosin Sl and tropomyosin from vertebrate striated muscle. The goals of the research proposed are to identify the actin moiety in Sl decorated filaments. To determine the magnitude and direction of the movement of tropomyosin during thin filament activation and to provide accurate maps of the decorated filament for future modelling studies with the high-resolution X-ray maps. To accomplish these goals, 3-D maps of F-actin and of thin filaments (actin, tropomyosin and the troponin complex) in the presence and absence of Ca2+ will be calculated and compared with each other and with already-completed maps of Sl decorated filaments. An adequate method to correct the data for the effects of the contrast transfer function of the microscope will be devised. The research will be undertaken according to the following plan: (1) Exploration of conditions for optimum preservation of the filaments. (2) Biochemical assessment to thin filament function under these conditions. (3) Collection, computer processing and averaging of data to provide 3-D maps of F-actin and thin filaments (+ and - Ca2+). (4) Comparison of these maps with already completed maps of the decorated filaments to: (i.) identify the actin moiety in decorated filaments (ii.) define the effect of Sl binding on the position of tropomyosin in the presence of Ca2+ (iii.) determine the magnitude of the movement of tropomyosin in thin filaments in response to Ca2+ binding by the troponin complex (iv.) explore the consequences of these findings for current models for Ca2+ mediated thin filament regulation of muscular contraction. (5) TMV will be used as a model system to explore the effects of the contrast transfer function. The techniques to be used have been applied successfully with Sl decorated filaments and include cryo-electron microscopy and computer processing and averaging of images.
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