Rosenfeld S S, Xing J, Cheung H C, Brown F, Kar S, Sweeney H L
Department of Neurology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 1998 Oct 30;273(44):28682-90. doi: 10.1074/jbc.273.44.28682.
In this study, we have examined the mechanism of phosphorylation-dependent regulation in smooth muscle myosin through the use of structural and kinetic methodologies applied to several myosin fragments. Fluorescence anisotropy decay measurements demonstrate that regulatory light chain phosphorylation significantly reduces the rotational correlation time of regulatable myosin preparations, whereas minimally regulated ones show little effect in this assay. Sedimentation equilibrium studies show that the regulatory domain can dimerize with a dissociation constant that is unaffected by regulatory light chain phosphorylation. Finally, kinetic studies on the interactions of myosin-ADP constructs with actin are also consistent with a model in which interactions occur between the two heads, which are lost with regulatory light chain phosphorylation. We propose that in the absence of regulatory light chain phosphorylation, the two heads of myosin interact with each other, due to a weak intrinsic dimerization of the regulatory domains that is significantly stabilized by the proximal rod. Regulatory light chain phosphorylation abolishes the stabilizing effect of the proximal rod, leading to a loss of this interaction.
在本研究中，我们通过对多个肌球蛋白片段应用结构和动力学方法，研究了平滑肌肌球蛋白中磷酸化依赖性调节的机制。荧光各向异性衰减测量表明，调节性轻链磷酸化显著降低了可调节肌球蛋白制剂的旋转相关时间，而在该测定中，调节程度最小的制剂几乎没有影响。沉降平衡研究表明，调节结构域可以以解离常数二聚化，该解离常数不受调节性轻链磷酸化的影响。最后，对肌球蛋白 - ADP构建体与肌动蛋白相互作用的动力学研究也与一个模型一致，在该模型中，两个头部之间发生相互作用，而这种相互作用在调节性轻链磷酸化时丧失。我们提出，在没有调节性轻链磷酸化的情况下，肌球蛋白的两个头部相互作用，这是由于调节结构域的弱内在二聚化，而近端杆显著稳定了这种二聚化。调节性轻链磷酸化消除了近端杆的稳定作用，导致这种相互作用丧失。
