Institute of Cytology of the Russian Academy of Sciences, Laboratory of Molecular Basis of Cell Motility, 4 Tikhoretsky Ave., 194064, Saint Petersburg, Russia; Saint Petersburg State University, Faculty of Biology, Department of Biophysics, 7/9 Universitetskaya Emb., 199034, Saint Petersburg, Russia.
Institute of Cytology of the Russian Academy of Sciences, Laboratory of Molecular Basis of Cell Motility, 4 Tikhoretsky Ave., 194064, Saint Petersburg, Russia.
Arch Biochem Biophys. 2018 Apr 15;644:17-28. doi: 10.1016/j.abb.2018.03.002. Epub 2018 Mar 3.
Using the polarized photometry technique we have studied the effects of two amino acid replacements, E240K and R244G, in tropomyosin (Tpm1.1) on the position of Tpm1.1 on troponin-free actin filaments and the spatial arrangement of actin monomers and myosin heads at various mimicked stages of the ATPase cycle in the ghost muscle fibres. E240 and R244 are located in the C-terminal, seventh actin-binding period, in f and b positions of the coiled-coil heptapeptide repeat. Actin, Tpm1.1, and myosin subfragment-1 (S1) were fluorescently labeled: 1.5-IAEDANS was attached to actin and S1, 5-IAF was bound to Tpm1.1. The labeled proteins were incorporated in the ghost muscle fibres and changes in polarized fluorescence during the ATPase cycle have been measured. It was found that during the ATPase cycle both mutant tropomyosins occupied a position close to the inner domain of actin. The relative amount of the myosin heads in the strongly-bound conformations and of the switched on actin monomers increased at mimicking different stages of the ATPase cycle. This might be one of the reasons for muscle dysfunction in congenital fibre type disproportion caused by the substitutions E240K and R244G in tropomyosin.
利用偏光光度法，我们研究了两个氨基酸替换（E240K 和 R244G）对肌球蛋白轻链 1.1（Tpm1.1）在肌球蛋白轻链 1.1 无肌钙蛋白肌动蛋白丝上的位置以及肌动蛋白单体和肌球蛋白头部的空间排列的影响在肌纤维幽灵中的 ATP 酶循环的各种模拟阶段。E240 和 R244 位于 C 末端，第七个肌动蛋白结合周期的 f 和 b 位置卷曲螺旋七肽重复。肌动蛋白、Tpm1.1 和肌球蛋白亚基 1（S1）用荧光标记：1.5-IAEDANS 附着在肌动蛋白和 S1 上，5-IAF 结合在 Tpm1.1 上。标记的蛋白质被掺入肌纤维幽灵中，并测量了 ATP 酶循环过程中偏振荧光的变化。结果发现，在 ATP 酶循环过程中，两种突变型原肌球蛋白都占据了靠近肌动蛋白内域的位置。在模拟不同的 ATP 酶循环阶段时，强烈结合构象的肌球蛋白头部的相对数量和处于开启状态的肌动蛋白单体的相对数量增加。这可能是原肌球蛋白中 E240K 和 R244G 取代引起的先天性纤维类型比例失调导致肌肉功能障碍的原因之一。
