TOPOGRAPHICAL STUDIES OF SMOOTH AND NONMUSCLE MYOSINS

平滑肌肌球蛋白和非肌肉肌球蛋白的形貌研究

• 批准号: 6154469
• 负责人: Christine R Cremo
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6154469
• 关键词: adenosinetriphosphatase; affinity labeling; animal tissue; chemical kinetics; conformation; crosslink; enzyme activity; enzyme mechanism; enzyme structure; fluorescence polarization; fluorescence spectrometry; fluorescent dye /probe; gel filtration chromatography; high performance liquid chromatography; intermolecular interaction; mass spectrometry; microfilaments; mutant; myosins; phosphorylation; photolysis; protein folding; protein purification; smooth muscle; synthetic peptide

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.

平滑肌和非肌肉肌球蛋白II的磷酸化是必需的， 激活收缩。调节光中的单个丝氨酸残基 肌球蛋白的两个“头部”结构域中的每一个的链被一个 Ca 2+激活激酶。目前的知识表明，这一戏剧性的 调节作用是通过大的构象变化介导的 肌球蛋白结构本提案的长期目标是确定 磷酸化依赖性调节机制的结构基础。 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的 调控基于这些和其他数据，提出了一个假设， 这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结构域。 假设：两个肌球蛋白头部结构域之间的相互作用是有利的， 非磷酸化的“关闭”状态，磷酸化的作用是 减少这些相互作用，从而使肌球蛋白采用力 在肌动蛋白存在的情况下产生状态。该委员会的具体目标 建议通过回答两个相关问题来测试该假设： l）肌球蛋白头之间的相互作用是否发生在 根据假设预测？ 2)肌球蛋白结构的哪些特定成分参与了 头与头之间的互动 为了实现特定的目标，荧光，光交联和铁- BDTA介导的肽切割实验将在孔- 定义的实验条件，旨在测试假设。 此外，小说的结构与活动之间的关系 一类新的工程非肌肉肌球蛋白，含有一个未修饰的 将研究一个完整的头部和一个部分头部，以具体实现 目标#2。