Mecahnism of the Conformational Change in E1E2-Type Transport Atpases

各种各样的生命过程，从肌肉收缩到营养吸收，都是由离子在细胞膜上的选择性运输触发或驱动的。在哺乳动物活性离子运输的几乎所有模型中，工作假设是载体可以呈现不同的构象，其中离子结合位点交替暴露在膜的相反两侧。构象变化的机理尚不清楚。构象变化是通道，还是涉及蛋白质结构的广泛重排？本申请中提出的研究的主要目的是获得反应机制中构象转变步骤的物理化学数据，以便在活性离子传输的备选方案中进行区分。通过荧光标记e1e2 -泵报告的构象变化的正向和反向速率将作为温度的函数进行测量。用绝对反应速率理论对结果进行分析，通过将决定反应速率和程度的能量分解为化学键形成或裂解引起的热变化，以及系统组织中增加或减少分子自由度的变化，对正在发生的分子化学事件的类型进行了限制。转运酶、一价阳离子和溶剂结构将根据经验变化，以了解哪些反应物是重要的，以及它们如何影响e1e2 -酶的构象变化。

A variety of life processes, ranging from muscle contraction to nutrient absorption, are either triggered or driven by the selective transport of ions across cell membranes. The working hypothesis in virtually all models of mammalian active ion transport is that the carrier can assume different conformations in which the ion binding sites are alternately exposed to opposite sides of the membrane. The mechanism of the conformational change is unknown. Does the conformational change gate a channel, or is extensive rearrangement of the protein structure involved? The principal objective of the research proposed in this application is to obtain physicochemical data on the conformational transition step in the reaction mechanism to permit discrimination among alternative proposals for active ion transport. The forward and reverse rates of the conformational change reported by fluorescent labeling of E1E2-pumps will be measured as a function of temperature. Analysis of the results by absolute reaction rate theory places contraints on the types of molecular chemical events that are occurring by resolving the enery which determines the rate and extent of reaction into heat changes resulting from formation, or cleavage, of chemical bonds and into changes in the organization of the system that increase, or decrease, the number of molecular degrees of freedom. The transport enzyme, the monovalent cation and the solvent structure will be empirically varied to learn which reactants are important and how they affect the conformational change in E1E2-enyzmes.