title: Three distinct actin-attached structural states of myosin in muscle fibers.
abstract: We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M'.D.P, we used pPDM to cross-link SH1 (Cys(707)) to SH2 (Cys(697)) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M'.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M'D.P analog on the force generation pathway.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Mello
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry* / Q000378:metabolism*; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D013314:Stress, Mechanical; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D005609:Free Radicals; D008301:Maleimides; D018994:Myosin Light Chains; D011758:Pyrroles; D013438:Sulfhydryl Compounds; C011328:N,N'-4-phenylenedimaleimide