Single-molecule tracking of myosins with genetically engineered amplifier domains

We combined protein engineering and single molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated artificial neck domains. Our results show that the step size has a clear linear dependence on the length of the neck domain and we also established that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step was ∼30°. The engineered change in the step size of myosin marks a significant advance in our ability to selectively modify the functional properties of molecular motors.

[1]  Justin E. Molloy,et al.  The motor protein myosin-I produces its working stroke in two steps , 1999, Nature.

[2]  J. Spudich,et al.  Single myosin molecule mechanics: piconewton forces and nanometre steps , 1994, Nature.

[3]  D. Manstein,et al.  Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain. , 1998, Biochemistry.

[4]  A. Houdusse,et al.  Structure of the regulatory domain of scallop myosin at 2 A resolution: implications for regulation. , 1996, Structure.

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

[6]  K C Holmes,et al.  Structural mechanism of muscle contraction. , 1999, Annual review of biochemistry.

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

[8]  E. Meyhöfer,et al.  The force generated by a single kinesin molecule against an elastic load. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Daniel Safer,et al.  Myosin VI is an actin-based motor that moves backwards , 1999, Nature.

[10]  H. Schuster,et al.  Cloning vectors for the production of proteins in Dictyostelium discoideum. , 1995, Gene.

[11]  D. Manstein,et al.  Kinetic characterization of a cytoplasmic myosin motor domain expressed in Dictyostelium discoideum. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[12]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

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

[14]  Toshio Yanagida,et al.  A single myosin head moves along an actin filament with regular steps of 5.3 nanometres , 1999, Nature.

[15]  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.

[16]  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.

[17]  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.

[18]  D A Parry,et al.  Analysis of the three-alpha-helix motif in the spectrin superfamily of proteins. , 1992, Biophysical journal.

[19]  W H Guilford,et al.  Two heads of myosin are better than one for generating force and motion. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. T. Tregear,et al.  Movement and force produced by a single myosin head , 1995, Nature.

[21]  A. Mehta,et al.  Detection of single-molecule interactions using correlated thermal diffusion. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[22]  W H Guilford,et al.  Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. , 1997, Biophysical journal.

[23]  Paul Young,et al.  Molecular Basis for Cross-Linking of Actin Filaments: Structure of the α-Actinin Rod , 1999, Cell.

[24]  A J Rowe,et al.  Association of structural repeats in the alpha-actinin rod domain. Alignment of inter-subunit interactions. , 1995, Journal of molecular biology.

[25]  M. Kollmar,et al.  Structure of a genetically engineered molecular motor , 2001, The EMBO journal.

[26]  D. Manstein,et al.  Dictyostelium discoideum myosin II: characterization of functional myosin motor fragments. , 1997, Biochemistry.

[27]  T. Yanagida,et al.  Multiple- and single-molecule analysis of the actomyosin motor by nanometer-piconewton manipulation with a microneedle: unitary steps and forces. , 1996, Biophysical journal.

[28]  M. Irving,et al.  Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction , 1999, Nature.

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