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bioRxiv · 10.1101/2025.05.01.651501

S2Tag, a novel affinity tag for the capture and immobilization of coiled-coil proteins: application to the study of human β-cardiac myosin

Barua, B.·Cail, R. C.·Goldman, Y. E.·Ostap, E. M.·Winkelmann, D. A.
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Abstract

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 {beta}-cardiac myosin construct (WT-{beta}CM) and evaluated its mechanochemistry. WT-{beta}CM 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 {beta}CM construct containing a penetrant mutation (S532P-{beta}CM) that causes dilated cardiomyopathy. Inclusion of S2Tag enabled quantitative mixed-motor gliding filament assays with WT-{beta}CM. 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-{beta}CM. Importantly, S2Tag is a useful new tool for affinity capture of alpha-helical coiled coil proteins.

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Barua, B., Cail, R. C., Goldman, Y. E., Ostap, E. M., Winkelmann, D. A.. 2025-05-02. S2Tag, a novel affinity tag for the capture and immobilization of coiled-coil proteins: application to the study of human β-cardiac myosin. https://doi.org/10.1101/2025.05.01.651501

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