The regulation of skeletal, cardiac and smooth muscle contraction involves a sequence of changing interaction within and between all of the thin filament proteins, and also of myosin. A major objective is to learn how the conformational properties of tropomyosin are related to its function. Previous several approaches were developed utilizing fluorescence and spin probes placed at a specific residue in tropomyosin's sequence to successfully probe conformational changes with tropomyosin. This approach also related conformational differences between smooth and striated tropomyosin to differences in activity. We plan to use these approaches to study conformational changes in tropomyosin in reconstituted thin filament systems with the aim of relating these differences to the """"""""on"""""""" and """"""""off"""""""" activity states of muscle. The binding of troponin and myosin sub-fragment 1 to the labeled tropomyosin-actin thin filament will allow differences in free energy between the different thin filament states to be determined. Rates of fluctuation between conformational states will be related to rates of switching on of muscle. The conformational properties of the different molecular forms of striated tropomyosin (Alpha Alpha, Alpha Beta, Beta) and near different residues along its sequence will be studied with suitably placed circular dichroism and fluorescence labels. In addition, differences between conformational properties of arterial smooth and striated tropomyosin will also be studied. Previously, studies were also begun on the conformational properties of portions of myosin rod which will be extended in the next grant period. Myosin road has a similar structure to tropomyosin including conveniently located Cys residues which make it amenable for labelling with fluorescence probes and interpreting experiments along the lines successfully used for tropomyosin. It is planned to relate the conformational properties of the myosin rod to possible roles in force generation.
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