title: Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation.
abstract: Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.
authors.affiliation: National High Magnetic Field Laboratory, 1800 East Paul Dirac Drive, Tallahassee, FL 32310, USA.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Vileno
authors.forename: Jean
authors.initials: J
authors.lastname: Chamoun
authors.forename: Hua
authors.initials: H
authors.lastname: Liang
authors.forename: Paul
authors.initials: P
authors.lastname: Brewer
authors.forename: Brian D
authors.initials: BD
authors.lastname: Haldeman
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Facemyer
authors.forename: Bridget
authors.initials: B
authors.lastname: Salzameda
authors.forename: Likai
authors.initials: L
authors.lastname: Song
authors.forename: Hui-Chun
authors.initials: HC
authors.lastname: Li
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
authors.forename: Piotr G
authors.initials: PG
authors.lastname: Fajer
mesh_terms: D000255:Adenosine Triphosphate; D000494:Allosteric Regulation; D000818:Animals; D002645:Chickens; D004578:Electron Spin Resonance Spectroscopy; D018994:Myosin Light Chains / Q000378:metabolism; D024462:Myosin Type II / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology*; D011487:Protein Conformation; D024745:Smooth Muscle Myosins / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D018994:Myosin Light Chains; D013113:Spin Labels; D000255:Adenosine Triphosphate; D024462:Myosin Type II; D024745:Smooth Muscle Myosins