title: Calcium-dependent interaction sites of tropomyosin on reconstituted muscle thin filaments with bound Myosin heads as studied by site-directed spin-labeling.
abstract: To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca(2+) ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca(2+). These findings suggest that even in the myosin-bound (open) state, Ca(2+) may regulate actomyosin contractile properties via Tm.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ueda
authors.forename: Chieko
authors.initials: C
authors.lastname: Kimura-Sakiyama
authors.forename: Tomoki
authors.initials: T
authors.lastname: Aihara
authors.forename: Masao
authors.initials: M
authors.lastname: Miki
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Arata
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013113:Spin Labels; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism
chemical_list: D000199:Actins; D013113:Spin Labels; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium