Mechanism of Thin Filament Regulation of Cardiac Actomyosin Hydrolysis

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DESCRIPTION (provided by applicant): We will use presteady state kinetic methods and electron microscopy to determine the mechanism of thin filament regulation of cardiac actomyosin ATP hydrolysis. Most biochemistry and physiology textbooks depict a "steric blocking" mechanism of thin filament regulation in which tropomyosin blocks the binding of myosin to actin at low calcium. However, there is now compelling experimental data that are inconsistent with this mechanism. Calcium and rigor myosin binding to fast skeletal muscle thin filaments accelerate the maximum rate of the product dissociation from myosin-ADP-Pi approximately 200 times but produce at most a 3 fold increase in the affinity of myosin-ADP-Pi binding to the thin filament. This indicates that steric blocking is a very small component of the regulatory mechanism. There are significant differences in the amino acid sequences between skeletal and cardiac actin, myosin, troponin, and tropomyosin and the mechanism of calcium regulation of cardiac actomyosin ATP hydrolysis is much less well characterized. Cardiac troponin- C has only a single regulatory calcium binding site and cardiac troponin-l has a 26 amino acid extension that contains a physiologically significant phosphorylation site. In addition to providing key information in the study of the mechanism of thin filament regulation, the proposed work is directly relevant to a number of diseases involving control of the regulation of cardiac muscle contraction, such as dilated cardiomyopathy, in which the force of the contraction is inadequate. Multi-mixing stopped-flow will be used to measure the dependence of the acceleration of product dissociation (Pi and ADP) from cardiac myosin-ADP-Pi by native cardiac thin filaments upon calcium and rigor myosin binding to the thin filament, and from phosphorylation of troponin-l. Parallel electron microscopy using negative stain and cryo-EM methods will be used to determine the structure of cardiac thin filaments (in various states of activation by calcium and bound myosin) and the distribution of the myosin bound to thin filaments. The structural data will be analyzed by single particle methods that have been adapted to filament structures by the laboratory of the CoPI (John Trinick). The combined data will be used to determine the mechanism of regulation by cardiac thin filaments and increase our understanding of mechanism of regulation of cardiac contractility.