Shchepkin Daniil V, Nabiev Salavat R, Kopylova Galina V, Matyushenko Alexander M, Levitsky Dmitrii I, Bershitsky Sergey Y, Tsaturyan Andrey K
Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, 620049, Russia.
A.N. Bach Institute of Biochemistry, Research Centre of Biotechnology, Russian Academy of Sciences, Moscow, 119071, Russia.
J Muscle Res Cell Motil. 2017 Apr;38(2):183-191. doi: 10.1007/s10974-017-9472-x. Epub 2017 May 24.
Muscle contraction is powered by myosin interaction with actin-based thin filaments containing Ca-regulatory proteins, tropomyosin and troponin. Coiled-coil tropomyosin molecules form a long helical strand that winds around actin filament and either shields actin from myosin binding or opens it. Non-canonical residues G126 and D137 in the central part of tropomyosin destabilize its coiled-coil structure. Their substitutions for canonical ones, G126R and D137L, increase structural stability and the velocity of sliding of reconstructed thin filaments along myosin coated surface. The effect of these stabilizing mutations on force of the actin-myosin interaction is unknown. It also remains unclear whether the stabilization affects single actin-myosin interactions or it modifies the cooperativity of the binding of myosin molecules to actin. We used an optical trap to measure the effects of the stabilization on step size, unitary force and duration of the interactions at low and high load and compared the results with those obtained in an in vitro motility assay. We found that significant prolongation of lifetime of the actin-myosin complex under high load observed at high extent of tropomyosin stabilization, i.e. with double mutant, G126R/D137L, correlates with higher force in the motility assay. Also, the higher the extent of stabilization of tropomyosin, the fewer myosin molecules are needed to propel the thin filaments. The data suggest that the effects of the stabilizing mutations in tropomyosin on the myosin interaction with regulated thin filaments are mainly realized via cooperative mechanisms by increasing the size of cooperative unit.
肌肉收缩由肌球蛋白与包含钙调节蛋白、原肌球蛋白和肌钙蛋白的基于肌动蛋白的细肌丝相互作用提供动力。卷曲螺旋的原肌球蛋白分子形成一条长螺旋链，缠绕在肌动蛋白丝周围，要么屏蔽肌动蛋白使其不与肌球蛋白结合，要么打开它。原肌球蛋白中部的非经典残基G126和D137使其卷曲螺旋结构不稳定。将它们替换为经典残基，即G126R和D137L，可增加结构稳定性以及重建的细肌丝沿肌球蛋白包被表面滑动的速度。这些稳定突变对肌动蛋白 - 肌球蛋白相互作用力的影响尚不清楚。同样不清楚的是，这种稳定性的增加是影响单个肌动蛋白 - 肌球蛋白相互作用，还是改变了肌球蛋白分子与肌动蛋白结合的协同性。我们使用光镊来测量这种稳定性对低负荷和高负荷下相互作用的步长单位力和持续时间的影响，并将结果与体外运动测定中获得的结果进行比较。我们发现，在高负荷下，当原肌球蛋白高度稳定时，即具有双突变体G126R/D137L时，观察到肌动蛋白 - 肌球蛋白复合物寿命显著延长，这与运动测定中的更高力相关。此外，原肌球蛋白的稳定程度越高，推动细肌丝所需的肌球蛋白分子就越少。数据表明，原肌球蛋白中稳定突变对肌球蛋白与受调节细肌丝相互作用的影响主要通过增加协同单位的大小，经由协同机制实现。
