title: Structural and functional effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin molecule.
abstract: Tropomyosin (Tm) is an α-helical coiled-coil protein that binds along the length of actin filament and plays an essential role in the regulation of muscle contraction. There are two highly conserved non-canonical residues in the middle part of the Tm molecule, Asp137 and Gly126, which are thought to impart conformational instability (flexibility) to this region of Tm which is considered crucial for its regulatory functions. It was shown previously that replacement of these residues by canonical ones (Leu substitution for Asp137 and Arg substitution for Gly126) results in stabilization of the coiled-coil in the middle of Tm and affects its regulatory function. Here we employed various methods to compare structural and functional features of Tm mutants carrying stabilizing substitutions Arg137Leu and Gly126Arg. Moreover, we for the first time analyzed the properties of Tm carrying both these substitutions within the same molecule. The results show that both substitutions similarly stabilize the Tm coiled-coil structure, and their combined action leads to further significant stabilization of the Tm molecule. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca(2+) concentrations but also increases Ca(2+) sensitivity of the actin-myosin interaction underlying this sliding. We propose that the effects of these substitutions on the Ca(2+)-regulated actin-myosin interaction can be accounted for not only by decreased flexibility of actin-bound Tm but also by their influence on the interactions between the middle part of Tm and certain sites of the myosin head.
authors.affiliation: A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Matyushenko
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Artemova
authors.forename: Daniil V
authors.initials: DV
authors.lastname: Shchepkin
authors.forename: Galina V
authors.initials: GV
authors.lastname: Kopylova
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Bershitsky
authors.forename: Andrey K
authors.initials: AK
authors.lastname: Tsaturyan
authors.forename: Nikolai N
authors.initials: NN
authors.lastname: Sluchanko
authors.forename: Dmitrii I
authors.initials: DI
authors.lastname: Levitsky
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D014335:Tropomyosin; D009218:Myosins; D002118:Calcium
keywords: Ca2+ regulation of muscle contraction; actin-myosin interaction; coiled-coil stabilization; flexibility; in vitro motility assay; tropomyosin