The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.
The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation.
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DOI：
10.1016/j.bpj.2015.06.038
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发表时间：
2015-08-18
影响因子：
3.4
通讯作者：
Irving M
中科院分区：
文献类型：
--
作者：
Fusi L;Huang Z;Irving M
在从许多类型的肌肉中分离出来的粗纤维中，每个肌凝蛋白分子的两个头部结构域被折叠回纤维骨架，形成一种称为相互作用头部基序（IHM）的构象，在这种构象中肌动蛋白的相互作用被抑制。这种构象存在于静止的骨骼肌中，但不知道在肌肉激活期间如何从IHM状态中退出。在这里，我们通过测量兔腰肌松弛脱膜纤维中肌球蛋白头轻链结构域的原位构象，使用荧光偏振的双功能罗丹明探针在肌球蛋白调节轻链（RLC）的c端叶的四个位点进行了研究。在松弛条件下，描述探针相对于丝轴取向的序参数< P2 >与温度大致呈s型关系，在~ 19°C时变化最大。用5%葡聚糖T500压缩晶格或添加25 μM blebbistatin可将转变温度降低至~ 14℃。最大熵分析显示，在25℃及以上温度下，肌球蛋白RLC区有三种优先取向，其中两种取向的长轴大致平行于丝轴，另一种取向的长轴大致垂直于丝轴。平行方向类似于IHM中所谓的阻塞头和自由头，并通过晶格压缩或blebbistatin来稳定。在接近生理温度和肌丝晶格间距的松弛骨骼肌中，大多数肌凝蛋白头部的轻链结构域呈ihm样构象，少数具有不同的构象，其RLC区域大致垂直于丝轴。这三种取向种群在活动收缩期间均不存在。这些结果与骨骼肌中粗丝的调节转变与肌凝蛋白头部的构象平衡相一致。
In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.