Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket

H. Lee Sweeney, Steven S. Rosenfeld, Fred Brown, Lynn Faust, Joe Smith, Jun Xing, Leonard A. Stein, James R. Sellers

Research output: Contribution to journalArticle

191 Scopus citations

Abstract

A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin has been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myosin filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To test this hypothesis, loops derived from different myosin II isoforms that display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced by baculovirus/Sf9 cell expression. Although the nature of this loop affected the rate of ADP release (up to 9- fold), in vitro motility (2.7-fold), and the V(max) of actin-activated ATPase activity (up to 2-fold), the properties of each chimera did not correlate with the relative speed of the myosin from which the loop was derived. Rather, the rate of ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament translocation was altered by the rate of ADP release, but was not solely determined by it. Through a combination of solute quenching and transient fluorescence measurements, it is concluded that, as the loop gets smaller, access to the nucleotide pocket is more restricted, ATP binding becomes less favored, and ADP binding becomes more favored. In addition, the rate of ATP hydrolysis is slowed.

Original languageEnglish (US)
Pages (from-to)6262-6270
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number11
DOIs
StatePublished - Mar 13 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Sweeney, H. L., Rosenfeld, S. S., Brown, F., Faust, L., Smith, J., Xing, J., Stein, L. A., & Sellers, J. R. (1998). Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket. Journal of Biological Chemistry, 273(11), 6262-6270. https://doi.org/10.1074/jbc.273.11.6262