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The effect of G126R and D137L mutations in α-tropomyosin on the position and flexibility of tropomyosin strands during the ATPase cycleтезисы доклада
- Авторы: Rysev Nikita A., Matyushenko Alexander M., Artemova Natalia V., Levitsky Dmitrii I., Borovikov Yurii S.
- Сборник: Materials of International Symposium "Biological motility: new facts and hypotheses "
- Тезисы
- Год издания: 2014
- Место издания: ООО "Фотон-век" Pushchino
- Первая страница: 230
- Последняя страница: 232
- Аннотация: Tropomyosin molecule (TM) is a semi-rigid structure which is stiff enough to move across the surface of actin as a unit when perturbed mechanically by other actin-binding proteins such as myosin. TM is a two chain α-helical coiled coil whose periodic interactions with the F-actin helix are critical for thin filament stabilization and the regulation of muscle contraction. It is believed that non-canonical residues Asp137 and Gly126 located in the middle part of TM molecule can destabilize the molecule at these points. Thus recently it has been shown that the Asp substitution for Leu at 137 position and the replacement of Gly126 with Ala or Arg significantly stabilize the middle part of the TM molecule [1, 2, 4]. This stabilization not only enhances maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca2+ concentrations but also increases Ca2+-sensitivity of the actin-myosin interaction underlying this sliding [3, 4]. To examine the effect of the substitution of Gly126 with an Arg residue or Asp137 with a Leu residue and of both substitutions within the same skeletal α-TM molecule on the position and flexibility of the N terminus of TM and the spatial arrangement of actin monomers during the ATPase cycle we labelled the recombinant wild type and mutant TMs with 5-IAF at Cys36 (which was introduced into the N-terminal part of TM by mutation C190A/S36C) and F-actin with FITC-phalloidin, incorporated them into troponin-free ghost muscle fibres and studied their polarized fluorescence at different stages of the ATPase cycle. In the absence of myosin subfragment 1 (S1), these mutations shift TM towards the periphery of the filament, dramatically enhance TM strands rigidity, and slightly increase a proportion of the switched-on subunits in F-actin. The binding of S1 to F-actin (in the absence or presence of MgADP) moves all mutant TMs further to the center of the filaments (towards the “open position”), which results in a decreased flexibility of the N-terminus of TM and a pronounced rotation of actin monomers to the periphery of the filaments. The latter indicates an increase in the number of the switched on actin monomers. Under conditions mimicking the weak-binding states, the mutant TMs move further to the periphery of the filament and the amount of the switched on actin monomers extremely decreases compared to that for the wild type TM. We suggest that the observed increase in Ca2+-sensitivity of actin-myosin interaction and maximal sliding velocity of actin filaments in the in vitro motility assay induced by Asp137Leu and Gly126Arg substitutions in TM [3, 4] may result from the enhanced efficiency of the cross-bridge work caused by the abnormal TM shift further towards the center and periphery of the filaments at the strong-binding and weak-binding states, respectively. This work was supported by RFBR (grants 14-04-00454-a, 12-04-00411-a, and 13-04-40099-H).
- Добавил в систему: Боровиков Юрий Сергеевич