Hypertrophic cardiomyopathy (HCM) is a symptomatic affliction due to mutations in cardiac contractile proteins. We compared a highly penetrant mutation in cardiac myosin, M493I, with wild type (WT) using an advanced optical trap assay capable of quantifying kinetic rates including actomyosin re-attachment. Kinetic changes in both the actomyosin attachment and detachment rates suggest that the equilibrium between a conformation termed the Interacting Head Motif (IHM) and freely available myosin heads is disturbed in M493I. This type of disruption has been hypothesized to lead to the toxic hypertrophy observed in patients.
Imprecision in protein positioning and instrument dead time have hampered efforts to measure macromolecular association rates in optical trap assays. Here, we combine several technical improvements to the three-bead optical trap assay, including precise protein deposition, enhanced stage stability by feedback, and improved data filtering. These enhancements allow us to precisely and reliably detect interactions between cardiac heavy meromyosin (cHMM) and actin and quantify attachment and reattachment rates. These studies providing insights into strain-dependence of the power stroke and a proposed transition from super-relaxed (SRX) to disordered relaxed (DRX) states, which is thought to be disrupted in human hypertrophic cardiomyopathy.
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