Bridge 1: Conformational Transitions in P-type ATPases

桥 1：P 型 ATP 酶中的构象转变

• 批准号: 9149303
• 负责人: ROBERT K. NAKAMOTO
• 金额: $ 11.52万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-10 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9149303
• 关键词: ATP phosphohydrolase; Address; Algorithms; Amino Acid Substitution; Antigens; Binding; Biochemical; Biological Assay; Ca(2+)-Transporting ATPase; Catalysis; Chemicals; Cryoelectron Microscopy; Cytoplasmic Tail; Electrophysiology (science); Fluorescence; Heterogeneity; Image; Ion Transport; Ions; Kinetics; Laboratories; Life; Measurement; Membrane Proteins; Methods; Molecular Conformation; Monitor; Na(+)-K(+)-Exchanging ATPase; Nucleotides; Oocytes; Pathway interactions; Phase; Phosphorylation; Proteins; Pump; Reaction; Research Personnel; Resolution; Roentgen Rays; Sarcoplasmic Reticulum; Series; Signal Transduction; Site; Skeletal Muscle; Structure; Tertiary Protein Structure; Testing; Thermodynamics; Transport Reaction; base; conformational conversion; electric field; molecular dynamics; mutant; non-Native; particle; protein complex; research study; simulation; single molecule; unnatural amino acids; voltage

The P-type (or E1E2) ATPases undergo an extraordinary series of conformational changes during the course of their highly efficient transport cycles. Detailed structural studies of the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) and the Na++K+-ATPase (Na/K pump) in conditions that put the proteins into different chemically-defined intermediate states of the reaction cycle revealed important changes in the tertiary structures of the proteins both in the three major cytoplasmic domains and the transmembrane segments that comprise the ion transport sites. In this proposal, we address the conformational dynamics of the SERCA and Na/K pumps as they transition between intermediate states throughout their transport cycle. We will carry out the following specific aims: (1) correlate the dynamics between conformational states in the Na/K pump produced by voltage using site-directed fluorimetry, including missense incorporation of fluorescent unnatural amino acids in oocyte expression; (2) carry out a series of double-mutant cycle analyses of SERCA based on the predictions of non-native contacts identified by the molecular dynamics string method simulations of the transitions between chemically defined states; and (3) use single particle cryo-EM to evaluate the conformational heterogeneity of both Na/K pump and SERCA in different conformational states that are in different chemically defined catalytic states or transitioning between states. To facilitate such structural studies, we will create conformational specific single antigen binders to help identify the different domains.

P型（或E1E2）ATPases在课程过程中经历了一系列构象变化 它们高效的运输周期。骨骼肌肌质的详细结构研究 网状Ca2+-ATPase（SERCA1A）和Na ++ K+-ATPase（Na/K Pump）在放置蛋白质的条件下 在反应周期的不同化学定义的中间状态中发现了重要的变化 三个主要细胞质结构域和跨膜的蛋白质的三级结构 构成离子运输位点的细分市场。在此提案中，我们解决了的构象动态 SERCA和Na/k在整个中间状态之间在其运输周期之间过渡时泵。 我们将执行以下特定目的：（1）将构象状态之间的动态相关联 使用位置定向的荧光法产生的Na/k泵，包括荧光的错义掺入 卵母细胞表达中的非天然氨基酸； （2）对SERCA进行一系列双突变周期分析 基于通过分子动力学弦方法模拟确定的非本地接触的预测 化学定义状态之间的过渡； （3）使用单个粒子冷冻EM评估 Na/k泵和Serca的构象异质性在不同的构象状态中 不同化学定义的催化状态或状态之间的过渡。为了促进此类结构研究， 我们将创建构象特定的单一抗原粘合剂，以帮助识别不同的域。