TOPOGRAPHICAL STUDIES OF SMOOTH AND NONMUSCLE MYOSINS

平滑肌和非肌肉肌球蛋白II需要磷酸化才能 激活收缩。监管灯光下的单一丝氨酸残基 肌球蛋白的两个“头部”结构域的每个链都被一种 Ca~(2+)激活激酶。目前的知识表明，这一戏剧性的 调控效应是通过大的构象变化来调节的。 肌球蛋白结构。这项提案的长期目标是确定 依赖磷酸化的调控机制的结构基础。 私家侦探最近证明了两个相同的头部 这种肌球蛋白的结构域是磷酸化依赖所必需的 监管。基于这些和其他数据，提出了一个假设， 它是由最近的三维结构模型 骨骼肌球蛋白和扇贝肌球蛋白的调节域。 假设：两个肌球蛋白头部结构域之间的相互作用在 非磷酸化的“关”状态，而磷酸化的作用是 减少这些相互作用，从而允许肌球蛋白产生作用力 在肌动蛋白存在的情况下产生状态。《公约》的具体目标 建议通过回答两个相关问题来检验这一假设： L)肌球蛋白头部之间的相互作用在以下条件下发生 假说所预言的？ 2)肌球蛋白结构中的哪些特定元件参与了 推定的头部-头部互动？ 为了达到特定的目的，荧光，光交联和铁- BDTA介导肽的切割实验将在良好的 定义了用来检验假设的实验条件。 此外，小说的结构和活性之间的关系 含有一种未修饰的新型工程化非肌肉肌球蛋白 将研究完整的头部和一个部分的头部，以具体履行 目标2.

Phosphorylation of smooth muscle and nonmuscle myosin II is required to activate contraction. A single serine residue in the regulatory light chain of each of the two "head" domains of myosin is phosphorylated by a Ca2+-activated kinase. The current knowledge suggests that this dramatic regulatory effect is mediated through large conformational changes in the myosin structure. The long term goal of this proposal is to determine the structural basis of the phosphorylation-dependent regulatory mechanism. The P.I. has recently demonstrated that both of the two identical head domains of this myosin are required for phosphorylation-dependent regulation. Based upon these and other data a hypothesis is proposed, which is guided by the recent three-dimensional structural model of skeletal myosin and the regulatory domain of scallop myosin. Hypothesis: Interaction between the two myosin head domains is favored in the unphosphorylated "off" state, and the role of phosphorylation is to diminish these interactions, thus allowing for myosin to adopt force generating states in the presence of actin. The specific aims of the proposal are to test the hypothesis by answering two related questions: l) Does an interaction between the myosin heads occur under conditions predicted by the hypothesis? 2) Which specific elements of the myosin structure are involved in the putative head-head interaction? To accomplish the specific aims, fluorescence, photocrosslinking and iron- BDTA mediated peptide cleavage experiments will be performed under well- defined experimental conditions which are designed to test the hypothesis. In addition, the relationship between structure and activity of a novel new class of engineered nonmuscle myosins that contain one unmodified complete head and one partial head will be studied to specifically fulfill aim #2.