FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

Laura K. Gunther, John A. Rohde, Wanjian Tang, Joseph A. Cirilo, Christopher P. Marang, Brent D. Scott, David D. Thomas, Edward P. Debold, Christopher M. Yengo

Research output: Contribution to journalReview articlepeer-review

Abstract

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Original languageEnglish (US)
Pages (from-to)17383-17397
Number of pages15
JournalJournal of Biological Chemistry
Volume295
Issue number51
DOIs
StatePublished - Dec 18 2020

Bibliographical note

Funding Information:
Funding and additional information—This work was supported by

Publisher Copyright:
© 2020 Gunther et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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