Dr. Laura Gunther Profile

Dr. Laura Gunther

at Penn Medicine

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Proceedings Article | 1 August 2021 Presentation + Paper

Using a single molecule optical trapping assay and FRET to reveal the mechanism of transduction by the molecular motor myosin

Edward Debold, Brent Scott, Chris Marang, Laura Gunther, Christopher Yengo

Proceedings Volume 11798, 1179804 (2021) https://doi.org/10.1117/12.2595724

KEYWORDS: Molecules, Optical tweezers, Fluorescence resonance energy transfer, Data modeling, Molecular lasers, Acquisition tracking and pointing, Silica, Proteins, Molecular interactions, Molecular energy transfer

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Myosin is biological molecular motor that transduces the chemical energy ATP hydrolysis into force and/or motion to drives many forms of cellular motility from muscle contraction to cell division. While the transduction process has been extensively studied key details remain unclear. Most importantly the timing of the force-generating powerstroke relative to the release of phosphate (Pi), a product of ATP hydrolysis, is not clear and the source of intense debate. We examined the ability of single-headed myosin Va to generate a displacement (i.e. powerstroke) of an actin filament in a kinetic, fluorescent energy transfer (FRET) and single molecule laser trap assays while maintaining Pi in its catalytic site by introducing a mutation that impedes Pi-release from the catalytic site (S217A). Kinetic experiments confirmed that the Pi-release rate was slowed ~5-fold by the S217A substitution, and FRET experiments suggested that the mutation slowed myosin’s rate of attachment to actin. At the single molecule level Wild type myosin Va (WT) generated a 7 nm powerstroke at a rate of ~600/s and the S217A myosin generated a statistically similar size powerstroke (8nm) at a similar rate (600/s) despite impeding the release of Pi from the active site. These findings are consistent with myosin Va generating a powerstroke while Pi is still in the catalytic site, and therefore challenge the hypothesis that Pi-release gates, and therefore precedes, the powerstroke. Our findings imply that myosin’s key mechanical event is triggered by binding to the actin filament, and not by the biochemical event of Pi-release. This provides important new insight into the mechanism of energy transduction by myosin, and evolutionarily related motor proteins.

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