title: Conformational changes at the nucleotide site in the presence of bound ADP do not set the velocity of fast Drosophila myosins.
abstract: The conformational changes in myosin associated with ADP release and their influence on actin sliding velocity are not understood. Following actin binding, the myosin active site is in equilibrium between a closed and open ADP bound state, with the open state previously thought to favor ADP release and thus expected to be favored in faster myosins. However, our recent work with a variety of myosins suggests the opposite, that the open conformation is dominant in slower myosins, which have higher ADP affinities. To test if this correlation holds for fast myosin isoforms, we determined the relationships between conformational pocket dynamics, ADP affinity and velocity of four Drosophila myosins: indirect flight muscle (IFM) myosin (IFI), embryonic muscle myosin (EMB) and two IFI/EMB chimeras. Electron paramagnetic resonance spectra of nucleotide-analog spin probes (SLADP) bound to IFI subfragment-1 in the absence of actin showed a high degree of immobilization, indicating a predominately closed nucleotide pocket. The A·M·SLADP spectra of all four myosins in fibers (actin bound) also indicated an equilibrium favoring the closed conformation with the closed state closing even further. However, the energetics of pocket closure did not correlate with Drosophila myosin actin velocity suggesting our previous model relating pocket dynamics to velocity does not hold for fast myosin isoforms. We conclude that for these fast myosins, and possibly other fast myosins, velocity is controlled by factors other than the ratio of open to closed nucleotide pocket conformation.
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Eldred
authors.forename: Nariman
authors.initials: N
authors.lastname: Naber
authors.forename: Edward
authors.initials: E
authors.lastname: Pate
authors.forename: Roger
authors.initials: R
authors.lastname: Cooke
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Swank
mesh_terms: D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004330:Drosophila / Q000166:cytology* / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D029721:Drosophila Proteins; D015879:Myosin Subfragments; D009711:Nucleotides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate