MOLECULAR ANALYSIS OF FHC MISSENSE MUTATIONS

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Structural models of how myosin moves actin and produces force hypothesize that the interface between the motor domains and the light-chain (LC) binding neck region is a pivot point for bending of the two domains relative to each other. Many point mutations implicated in familial hypertrophic cardiomyopathies (FHC) are clustered in the beta-myosin heavy chain at the essential LC (ELC) binding interface, as well as in the LCs themselves. The effect of heavy chain mutations found in FHC on myosin's enzymatic and mechanical properties (steady-state and transient kinetics, velocity, unitary force, and unitary step size) will be analyzed in the first aim. Heavy chain mutations will be engineered into smooth muscle myosin and expressed in the baculovirus/insect cell system, to determine which mutations are critical for the function of all myosins. A concurrent goal is to increase the yield of expressed cardiac HMM so that comparable experiments can be performed on expressed cardiac mutants. Cardiac myosin with a subset of these FHC mutations will also be isolated from transgenic mice (obtained from Core C), and analyzed similarly. Such comparative studies should contribute to understanding the basic mechanical properties of all myosins, as well as providing a molecular basis for the effect of selected FHC mutations. The second aim focuses on how FHC mutations in the cardiac regulatory and essential LCs affect cardiac myosin's enzymatic and mechanical properties. Since atrial ELC accumulates in the ventricle in different forms of human ventricular hypertrophies, the kinetic and mechanical properties of a beta-cardiac myosin/atrial ELC chimera will also be determined. In both cases, bacterially expressed cardiac LCs will be reconstituted with LC-deficient beta-cardiac myosin heavy chain prepared from tissue, and assayed for functional properties by the techniques described for the first aim. The overall goal of the proposal is to elucidate how mutations implicated in FHC affect the mechanical performance of myosin.