Laboratory of Mechanisms of Cell Motility, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
Arch Biochem Biophys. 2014 Feb 1;543:57-66. doi: 10.1016/j.abb.2013.12.016. Epub 2013 Dec 25.
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.
为了研究 Gly126Arg 突变在骨骼肌 α-TM 或平滑肌 β-TM 中诱导的 TM 稳定如何影响 TM 的柔韧性及其在无肌钙蛋白薄丝上的位置，我们用 5-IAF 标记重组野生型和突变型 TM，并将其与 FITC-鬼笔环肽标记的 F-肌动蛋白一起掺入到幽灵肌纤维中，并在 ATP 酶循环的不同阶段研究偏振荧光。已经表明，在肌球蛋白和肌钙蛋白缺失的丝中，Gly126Arg 突变导致 TM 链向肌动蛋白的外域移动，减少了处于激活状态的肌动蛋白单体的数量，并降低了α-TM C 端的刚性并增加了 β-TMs N 端的刚性。肌球蛋白亚基 1 的结合将野生型 TM 移向肌动蛋白的内域，降低了 TM 两端的柔韧性，并增加了处于激活状态的肌动蛋白单体的数量。在 ATP 酶循环过程中，观察到丝中的 α-和 β-TM 以及肌动蛋白单体的位置以及 TM 和 F-肌动蛋白的柔韧性发生了多步改变。Gly126Arg 突变使 TM 位置与丝中处于激活状态的肌动蛋白单体的数量之间的相关性脱耦。
