MYOSIN PHOSPHORYLATION BY MYOSIN LIGHT CHAIN KINASE

该项目旨在了解分子机制， 钙调蛋白（CaM）调节肌球蛋白轻链的酶活性 链激酶（MLCK），并导致钙依赖性 平滑肌肌球蛋白的磷酸化/活化。具体来说， 本课题主要研究了以下几个问题：（1）构象 兔平滑肌的催化/调节结构域的转换 MLCK通过其与CaM和肌球蛋白调节因子的相互作用诱导 轻链（RLC）。乳鼠体内抑制机制假说 将通过测量共振能量转移 位于天然MLCK和MLCK中关键位置的发色团 专门设计的突变体区内标志性地点之间的距离 将测量MLCK、CaM和RLC的二元和三元复合物。的 核苷酸和核苷酸类似物对这种复合物的影响将是 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位点将被 鉴定N-末端的结构和功能性质 哺乳动物平滑肌MLCK特异性的重复片段将是 表征了(3)MLCK在平滑肌细胞中的原位定位。 MLCK在不同条件下与其他蛋白质组分的分布 收缩条件将通过免疫电子显微镜来确定 使用对MLCK的不同片段特异的抗体。

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.