Immobilization of human β-cardiac myosin using the novel S2Tag revealed that the S532P dilated cardiomyopathy mutation results in a 60% decrease in gliding speed compared to wild-type.
The S2Tag is a novel and effective tool for the affinity capture and immobilization of alpha-helical coiled-coil proteins like human β-cardiac myosin for in vitro assays.
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.
Barua et al. (Mon,) conducted a other in Dilated cardiomyopathy (in vitro model). S2Tag vs. Wild-type β-cardiac myosin was evaluated on Mechanochemistry (gliding speed and force production). Immobilization of human β-cardiac myosin using the novel S2Tag revealed that the S532P dilated cardiomyopathy mutation results in a 60% decrease in gliding speed compared to wild-type.