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** and
- James R. Sellers‡
- From the ‡Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, the Departments of ¶Neurology and ‖Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, the **Department of Medicine, State University of New York, Stony Brook, New York 11794, and the‡Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
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 maxof 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.
Footnotes
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↵* This work was supported by National Institutes of Health NIAMSD Grant AR-35661.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Physiology, University of Pennsylvania School of Medicine, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. Tel.: 215-898-0485; Fax: 215-898-0475; E-mail: lsweeney{at}mail.med.upenn.edu.
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↵1 The abbreviations used are: S1, myosin subfragment 1; HMM, heavy meromyosin; mant, methylanthraniloyl; DTT, dithiothreitol; MOPS, 4-morpholinepropanesulfonic acid; ELC, essential light chain; RLC, regulatory light chain.
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↵2 S. S. Rosenfeld, J. Xing, H. Cheung, F. Brown, S. Kar, and H. L. Sweeney (1998), manuscript submitted.
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- Received October 10, 1997.
- Revision received December 22, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
