Based upon numerous reports of disease-causing missense mutations that alter protein folding, we hypothesize that the folding and/or mechanical stability of myosin and its essential and regulatory light chains (LCs) are also affected by mutations. We propose an interdisciplinary collaboration to (i) define the "mechanical fingerprint" of myosin's motor domain and (ii) test the hypothesis that highly pathogenic mutations in the light chains that cause hypertrophic cardiomyopathy (HCM) affect the folding and/or mechanical stability of the lever-arm complex. Most biochemical and biophysical studies of myosin have focused on non-human and non-cardiac isoforms given the difficulty in producing active human myosin and mutants. The Leinwand lab overcame these challenges to pioneer the kinetic description of disease-causing mutations of human beta-cardiac myosin. Prior single-molecule mechanical studies of rabbit skeletal muscle myosin by atomic force microscopy (AFM) underscored the important contribution of the muscle-specific chaperone, Unc45b. Yet, this assay lacked the sensitivity to resolve any unfolding fingerprint of individual myosin motor domains (and had a very low yield). By merging the Leinwand lab's expertise in the genetic, cellular, and biochemical origins of cardiomyopathy with the Perkins lab's technical advances in single-molecule AFM studies, we will characterize the unfolding intermediates of human wild-type beta-cardiac myosin motor domain and thereby establish its mechanical fingerprint, the foundation to interpreting disease-causing mutations effect on the folding and mechanical stability. Proof-of-principle data shows that these studies, carried out with a ~75-fold improvement in time resolution and ~10-fold improvement in force precision, reveal structural details of the motor domain unfolding for the first time. Mechanical stability is equally critical on smaller scales; the LCs rigidify myosin's lever arm. Using AFM, we will characterize the LCs and disease-causing mutation therein interacting with myosin's lever arm. Such single-molecule studies will be complemented by quantitative imaging of tagged wild-type and mutant LCs in beating cardiac myocytes, where preliminary data on 2 HCM mutations in the RLC show reduced binding to the sarcomere. In summary, the proposed work will provide a quantitative foundation to test therapeutic chaperones as potential drugs for disease- causing mutations in myosin and its associated LCs.
|Program type||Transformational Project Award|
|Effective start/end date||07/01/2018 → 06/30/2021|