Key result
S532P mutant β-cardiac myosin reduces gliding speed ~60% vs wild-type despite producing similar force.
Why the study?
Quality and reproducibility of myosin motility assay data depend critically on the mode of attachment of myosin to assay surfaces.
S2Tag is a novel affinity tag that enables effective immobilization of coiled-coil proteins like human β-cardiac myosin for ensemble and single-molecule assays, revealing that the S532P dilated cardiomyopathy mutation decreases gliding speed by 60% without affecting force production.
Findings in animal models should not yet alter dilated cardiomyopathy management; extends mechanistic insight into S532P myosin effects.
Single molecule and ensemble motility assays are powerful tools for investigating myosin activity. A key requirement for the quality and reproducibility of the data obtained with these methods is the mode of attachment of myosin to assay surfaces. We previously characterized the ability of a monoclonal antibody (10F12.3) to tether skeletal muscle myosin to nitrocellulose coated glass. Here, we identify the 11 amino-acid epitope (S2Tag) recognized by 10F12.3 in the coiled-coil S2 domain of myosin. To test the transferability of S2Tag, we inserted it into a wild-type β-cardiac myosin construct (WT-βCM) and evaluated its mechanochemistry. WT-βCM immobilized via S2Tag robustly propelled actin filaments in gliding assays and showed single-molecule actin displacements and attachment kinetics by optical trapping. Thus, the antibody attachment is effective for ensemble and single molecule assays. We inserted the S2Tag into a βCM construct containing a penetrant mutation (S532P-βCM) that causes dilated cardiomyopathy. Inclusion of S2Tag enabled quantitative mixed-motor gliding filament assays with WT-βCM. The analysis shows the S532P mutation results in a 60% decrease in gliding speed, yet the motor seems to produce the same force as WT-βCM. Importantly, S2Tag is a useful new tool for affinity capture of alpha-helical coiled coil proteins.
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Barua et al. (2025) studied Dilated cardiomyopathy (in vitro model). S2Tag insertion vs. Wild-type β-cardiac myosin was evaluated on Gliding speed and force production. Insertion of the S2Tag into a β-cardiac myosin construct with the S532P mutation revealed a 60% decrease in gliding speed compared to wild-type, despite producing similar force.
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