PROJECT SUMMARY Mutations in the human ?-cardiac myosin gene (MYH7) are responsible for a large number of inherited Hypertrophic (HCM) and Dilated Cardiomyopathies (DCM). The objective of the proposal is the biochemical and biophysical characterization of the effects of human cardiac myosin mutations as an essential first step in identifying the changes in the structure and mechanism that culminate in cardiomyopathies. A major hurdle in tackling this problem with human cardiac myosin has been the instability and heterogeneity of the protein obtained from patient tissues, and the lack of an adequate expression system to produce high quality human ?-cardiac myosin. We developed a mammalian expression system based on adenoviral infection of a muscle cell line that is now widely accepted as the model for these studies. This approach produces the quantities of the human ?-cardiac myosin required for detailed kinetic and structural studies. The crystal structures of the human ?-cardiac myosin motor domain reveal a cluster of HCM and DCM mutations in a region linking structural elements that are critical for mechanochemical coupling. We have called this the coupling region. Furthermore, we have shown that the cardiac myosin activator, omecamtiv mecarbil, binds in a narrow cleft in the center of the coupling region and acts by influencing the mechanochemical coupling mechanism. We propose to study eight HCM/DCM mutations with severe clinical phenotypes residing within the coupling region to quantify the effects on structure and mechanism of the mutations by: (1) steady-state and transient kinetic assays to quantify subtle changes in the catalytic mechanism; (2) motility assays to evaluate ensemble motor dynamics, and single molecule force measurements to measure unitary mechanical characteristics; and (3) crystallographic structure analysis and modeling to complement the biomechanical measurements and guide interpretation. The effect of omecamtiv mecarbil on the coupling mechanism of mutated cardiac myosin will provide insights into the potential for clinical management of the disease. Understanding the molecular basis of mechanical changes resulting from cardiac myosin mutations will aid in the development of therapeutic approaches to mitigate the dysfunction leading to cardiomyopathies.