Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

[1]  R. Cooke,et al.  The mechanism of muscle contraction. , 1986, CRC critical reviews in biochemistry.

[2]  G. Piazzesi,et al.  Elastic bending and active tilting of myosin heads during muscle contraction , 1998, Nature.

[3]  H E Huxley,et al.  The Mechanism of Muscular Contraction , 1965, Scientific American.

[4]  I. Schlichting,et al.  Structure of the regulatory domain of scallop myosin at 2.8 Ä resolution , 1994, Nature.

[5]  K. Trybus,et al.  Myosin conformational states determined by single fluorophore polarization. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Michael Whittaker,et al.  A 35-Å movement of smooth muscle myosin on ADP release , 1995, Nature.

[8]  Malcolm Irving,et al.  Tilting of the light-chain region of myosin during step length changes and active force generation in skeletal muscle , 1995, Nature.

[9]  A. Szent-Györgyi,et al.  Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects. , 1980, Journal of molecular biology.

[10]  R. Cooke,et al.  Orientation of spin labels attached to cross-bridges in contracting muscle fibres , 1982, Nature.

[11]  Hiroto Tanaka,et al.  Simultaneous Observation of Individual ATPase and Mechanical Events by a Single Myosin Molecule during Interaction with Actin , 1998, Cell.

[12]  D. D. Thomas,et al.  A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Cooke,et al.  Actomyosin interaction in striated muscle. , 1997, Physiological reviews.

[14]  M. Irving,et al.  Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle. , 1996, Biophysical journal.

[15]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[16]  Y. Goldman,et al.  Wag the Tail: Structural Dynamics of Actomyosin , 1998, Cell.

[17]  J. Spudich,et al.  The neck region of the myosin motor domain acts as a lever arm to generate movement. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Irving,et al.  Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres. , 1998, Journal of molecular biology.

[19]  A. Penzkofer,et al.  ORIENTATION OF TRANSITION DIPOLE MOMENTS OF RHODAMINE 6G DETERMINED BY EXCITED STATE ABSORPTION , 1980 .

[20]  G. Piazzesi,et al.  The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin. , 1998, Biophysical journal.

[21]  R A Milligan,et al.  Structure of the actin-myosin complex and its implications for muscle contraction. , 1993, Science.

[22]  M. Irving,et al.  Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine. , 1998, Biophysical journal.

[23]  T. Rowe,et al.  Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function. , 1992, The EMBO journal.

[24]  J. Corrie,et al.  A homobifunctional rhodamine for labeling proteins with defined orientations of a fluorophore. , 1998, Bioconjugate chemistry.

[25]  A. Huxley,et al.  Proposed Mechanism of Force Generation in Striated Muscle , 1971, Nature.

[26]  Roberto Dominguez,et al.  Crystal Structure of a Vertebrate Smooth Muscle Myosin Motor Domain and Its Complex with the Essential Light Chain Visualization of the Pre–Power Stroke State , 1998, Cell.

[27]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[28]  G. Piazzesi,et al.  Myosin head movements are synchronous with the elementary force-generating process in muscle , 1992, Nature.

[29]  William H. Press,et al.  Book-Review - Numerical Recipes in Pascal - the Art of Scientific Computing , 1989 .

[30]  Control of tension development in scallop muscle fibres with foreign regulatory light chains , 1980, Nature.

[31]  W. Kabsch,et al.  Atomic structure of the actin: DNase I complex , 1990, Nature.

[32]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[33]  R. Moss,et al.  Physiological effects accompanying the removal of myosin LC2 from skinned skeletal muscle fibers. , 1982, The Journal of biological chemistry.

[34]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[35]  M. Irving,et al.  Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers. , 1999, Biophysical journal.

[36]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.

[37]  G. Piazzesi,et al.  Elastic distortion of myosin heads and repriming of the working stroke in muscle , 1995, Nature.

[38]  K. Holmes,et al.  Induced Changes in Orientation of the Cross-Bridges of Glycerinated Insect Flight Muscle , 1965, Nature.

[39]  A. Huxley.  Muscular contraction. Review lecture , 1974 .

[40]  M Anson,et al.  Myosin motors with artificial lever arms. , 1996, The EMBO journal.

[41]  M. Irving,et al.  Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers. , 1998, Biophysical journal.

[42]  K. Holmes.  The swinging lever-arm hypothesis of muscle contraction , 1997, Current Biology.

[43]  D A Winkelmann,et al.  Three-dimensional structure of myosin subfragment-1: a molecular motor. , 1993, Science.