Current Research and Scholarly Interests
Mutations in the beta-cardiac myosin (a molecular motor) cause disastrous effects by manifestation of hypertrophic and dilated cardiomyopathy, a leading cause of cardiac death. We hypothesize that the such mutations cause fundamental mechanistic changes in the motor which in turn affect the efficiency of the motor in several ways. My current research involves single molecule enzyme kinetics and force measurements to link intrinsic changes in motor function to the various clinical outcomes.
Over 250 different single point mutations in the beta-cardiac myosin heavy chain can cause either hypertrophic or dilated cardiomyopathy (HCM or DCM), but the underlying molecular effects on the myosin molecule remain elusive. The primary reason the effect of HCM and DCM mutations on purified human beta-cardiac myosin has not been elucidated is due to difficulties in expressing this protein in a functional and highly purified form. This limitation has now been eliminated by the expression of human beta-cardiac myosin in a mammalian cell line. The Spudich lab have pioneered both in vitro motility assays and single molecule techniques to assess the function of mechanoenzymes such as myosin. The lab has now developed a feedback controlled dual beam optical trap assay to measure the stroke size under low load and the maximum force that single molecules of myosin can produce. My project involves development and utilization of laser trap single molecule assays to measure these parameters using expressed and purified human beta-cardiac myosin. We hypothesize that HCM and DCM mutations affect the underlying force producing capability of human beta-cardiac myosin in different ways and that the eventual clinical phenotypes of HCM and DCM are a result of a particular fundamental mechanistic change in the human beta-cardiac myosin. As a working hypothesis, we propose that mutations that lead to increased force production lead to HCM while those that lead to decreased force production lead to DCM. Mechanisms that lead to decreased and increased force production by myosin can be varied. The inherent force producing capability of the motor, for example, could be increased or decreased by mutations that change the spring constant of the elastic element of the motor. On the other hand, the force-producing capability of the sarcomere could be changed in either direction by changes in the duty ratio of the myosin (the fraction of the ATPase cycle that the head is strongly bound to actin). I plan to study a selected group of 10 mutations, in terms of determining the biomechanical parameters of wild type human alpha- and beta-cardiac myosin and of HCM and DCM mutant human beta-cardiac myosins with respect to their velocities at ~zero load, their duty ratios, their stroke sizes, and the maximum force they produce upon interacting with actin. I plan to extend these studies with physiologically relevant muscle system which is composed of actin, tropomyosin and troponin complexes.