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Structural Rearrangements in the Active Site of Smooth-Muscle Myosin

Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont

Correspondence: Address reprint requests to Christopher L. Berger, Dept. of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT 05405. Tel.: 802-656-0832; Fax: 802-656-0747; E-mail: cberger{at}uvm.edu.

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 motor domain essential light chain, which contains a single tryptophan (344W) located on a short loop between two alpha helixes that traverse the upper-50 kD subdomain in front of the active site. Fluorescence resonance energy transfer was examined between the 344W donor probe and 2'(3')-O-(N-methylanthraniloyl) (mant)-nucleotide acceptor probes in the active site of this construct. The observed fluorescence resonance energy transfer 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 an 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.

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