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Biophys. J. BioFAST: First Published June 10, 2005. doi:10.1529/biophysj.105.059840
© 2005 by the Biophysical Society.

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MUSCLE AND CONTRACTILITY

Structural Rearrangements in the Active Site of Smooth-Muscle Myosin

C. Ian Robertson 1, Donald P. Gaffney II 1, Lynn R. Chrin 1 and Christopher Lewis Berger 1*

1 University of Vermont

* To whom correspondence should be addressed. E-mail: christopher.berger{at}uvm.edu.

Submitted on January 19, 2005
Revised on February 25, 2005
Accepted on 25 May 2005


  Abstract
Structural rearrangements of the myosin upper 50 kD subdomain are thought to play a key role in coordinating actin binding with nucleotide hydrolysis during the myosin ATPase cycle. Such rearrangements could open and close the active site in opposition to the actin-binding cleft, helping explain the opposing affinities of myosin for actin and nucleotide. To directly examine conformational changes across the active site during the ATPase cycle we have genetically engineered a mutant of chicken smooth-muscle myosin, F344W-MDE, which contains a single tryptophan (344W) located on a short loop between two alpha helixes which traverse the upper 50 kD subdomain in front of the active site. Fluorescence resonance energy transfer (FRET) was examined between the 344W donor probe and mant-nucleotide acceptor probes in the active site of this construct. The observed FRET efficiencies were 6.4% in the presence of mant ADP and 23.8% in the presence of mant ATP, corresponding to distances of 33.4 Å and 24.9 Å, respectively. Our results are consistent with structural rearrangements in which there is a 8.5 Å closure between the 344W residue and the mant moiety during the transition from the strongly (ADP) to weakly (ATP) actin-bound states of the myosin ATPase cycle.

Key Words: ATPase, FRET, fluorescence spectroscopy, muscle contraction, myosin, protein structure




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Copyright © 2005 by the Biophysical Society.