title: Gly126Arg substitution causes anomalous behaviour of α-skeletal and β-smooth tropomyosins during the ATPase cycle.
abstract: To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle β-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of β-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and β-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.
authors.affiliation: Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
authors.forename: Nikita A
authors.initials: NA
authors.lastname: Rysev
authors.affiliation: Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
authors.forename: Ilya A
authors.initials: IA
authors.lastname: Nevzorov
authors.affiliation: Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
authors.forename: Stanislava V
authors.initials: SV
authors.lastname: Avrova
authors.affiliation: Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
authors.forename: Olga E
authors.initials: OE
authors.lastname: Karpicheva
authors.affiliation: Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Redwood
authors.affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia.
authors.forename: Dmitrii I
authors.initials: DI
authors.lastname: Levitsky
authors.affiliation: Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia. Electronic address: borovikov@incras.ru.
authors.forename: Yurii S
authors.initials: YS
authors.lastname: Borovikov
mesh_terms: D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D019943:Amino Acid Substitution; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D018482:Muscle, Skeletal / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation; D010590:Phalloidine / Q000378:metabolism; D017433:Protein Structure, Secondary; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000199:Actins; D005452:Fluoresceins; D014335:Tropomyosin; D010590:Phalloidine; C015296:5-iodoacetamidofluorescein; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate
keywords: F-actin; Fluorescence polarization; Ghost muscle fibres; Myosin; Tropomyosin; Troponin