MYOSIN PHOSPHORYLATION BY MYOSIN LIGHT CHAIN KINASE

该项目旨在了解分子机制，通过它 钙调素(CaM)调节肌球蛋白光的酶活性 链激酶(MLCK)，导致钙依赖 肌球蛋白的磷酸化/激活。具体地说，这 本课题主要研究了以下几个问题：(1)构象 兔血管平滑肌催化/调节结构域的转变 与CaM及肌球蛋白调节蛋白相互作用诱导的MLCK 轻链(RLC)。空间位内抑制机制假说 将被测试的是测量共振能量传递之间的距离 在原生MLCK中和在 特别设计的突变体。地标性建筑之间的距离 将测量MLCK、CaM和RLC的二元和三元络合物。这个 核苷酸和核苷酸类似物对这种复合体的影响将是 已评估。(2)MLCK片段在催化外的功能作用 核心。与肌动蛋白和肌球蛋白重链相互作用的部位 已确认身份。N-末端的结构和功能性质 哺乳动物平滑肌特异性的重复片段MLCK将是 特色化的。(3)MLCK在血管内皮细胞的原位定位。 MLCK与其他蛋白质组分在不同条件下的分布 收缩条件将通过免疫电子显微镜来确定。 使用针对MLCK不同片段的抗体。

This project is aimed at understanding the molecular mechanisms by which calmodulin (CaM) modulates the enzymatic activity of the myosin light chain kinase (MLCK) and leads to the calcium dependent phosphorylation/activation of smooth muscle myosin. Specifically, this project is focused on the following problems: (1) The conformational transitions in the catalytic/regulatory domain of rabbit smooth muscle MLCK induced by its interaction with CaM and with the myosin regulatory light chain (RLC). The hypothesis of the intrasteric inhibitory mechanism will be tested be measuring the distance between resonance energy transfer chromophores placed at key positions in the native MLCK and in specifically designed mutants. Distances between landmark sites in the binary and ternary complexes of MLCK, CaM and RLC will be measured. The effects of nucleotides and nucleotide analogues on such complexes will be evaluated. (2) The functional role of MLCK segments outside the catalytic core. Sites interacting with actin and myosin heavy chain will be identified. The structural and functional properties of the N-terminal repetitive segment specific for the mammalian smooth muscle MLCK will be characterized. (3) In situ localization of MLCK in the smooth muscle cell. Distribution of MLCK in relation to other protein components under various contractile conditions will be determined by immunoelectron microscopy using antibodies specific to various segments of MLCK.