Bridge 1: Conformational Transitions in P-type ATPases

桥 1：P 型 ATP 酶中的构象转变

基本信息

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
项目类别：
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

项目摘要

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）ATP 酶在此过程中经历一系列非凡的构象变化其高效的运输周期。骨骼肌肌浆的详细结构研究网状 Ca2+-ATP 酶 (SERCA1a) 和 Na++K+-ATP 酶（Na/K 泵）处于放置蛋白质的条件下进入反应循环的不同化学定义的中间状态揭示了重要的变化三个主要细胞质结构域和跨膜中蛋白质的三级结构组成离子传输位点的片段。在这个提案中，我们解决了构象动力学 SERCA 和 Na/K 泵在整个运输周期中在中间状态之间转换。我们将实现以下具体目标：（1）关联构象状态之间的动态使用定点荧光测定法通过电压产生 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) 进行SERCA的一系列双突变体循环分析基于分子动力学弦法模拟识别的非自然接触的预测化学定义状态之间的转变； (3) 使用单粒子冷冻电镜来评估 Na/K泵和SERCA在不同构象状态下的构象异质性不同化学定义的催化状态或状态之间的转变。为了促进此类结构研究，我们将创建构象特异性单抗原结合物来帮助识别不同的结构域。