Pilot 1: Investigating Conformational Changes in Saccharide Transporter

试点 1：研究糖转运蛋白的构象变化

基本信息

批准号：
8933663
负责人：
Ming Zhou
金额：
$ 26.55万
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 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8933663
关键词：
ATP Hydrolysis Active Sites Bacillus cereus Bacteria Binding Binding Sites Carbohydrates Carrier Proteins Cell membrane Cells Complex Computational Technique Computer Simulation Core Facility Coupled Cysteine Cytoplasm Cytosol Data Dimerization Docking Electrons Energy Transfer Enzymes Face Family Fluorescence Fostering Free Energy Glucose Transporter Goals Homologous Gene Integral Membrane Protein Lanthanoid Series Elements Mediating Membrane Membrane Proteins Metabolism Methodology Methods Molecular Molecular Conformation Motion Mutation Orthologous Gene Pathway interactions Phosphoenolpyruvate Phosphorylation Phosphotransferases Pilot Projects Process Protomer Reaction Resolution Resources Roentgen Rays Sampling Side Structural Models Structure Substrate Specificity System Techniques Testing X-Ray Crystallography base conformational conversion design extracellular inorganic phosphate member molecular dynamics periplasm phosphocarrier protein HPr prevent simulation single molecule sugar uptake

项目摘要

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates concentrative uptake of sugars across the inner membrane in bacteria by a unique mechanism, using phosphorylation of the sugar substrate in concert with transport to drive uptake against the concentration gradient. The PTS comprises several soluble phosphotransferases and an integral membrane protein, EIIC. The EIIC component is responsible for substrate specificity, translocation across the membrane, and is necessary for the phosphorylation reaction to proceed. We recently solved the X-ray structure of an EIIC homolog, ChbC, in an inward-occluded conformation, which has allowed us to propose hypotheses for the conformational changes that bring the substrate across the bilayer and for how the phosphorylation reaction occurs. Using the unique resources and expertise provided by the member labs and core facilities of the MPSDC, we intend to use a variety of structural, spectroscopic, and computational techniques to test these hypotheses and build a comprehensive model of the structural dynamics of the EIIC transport cycle. Specifically, we will combine X-ray crystallography, DEER EPR, smFRET, and molecular dynamics simulations to determine the structures of the outward-open and inward-open conformations of the transporter, and to identify how EIIC forms a ternary complex with its sugar substrate and the soluble PTS component EIIB in order to catalyze the phosphorylation reaction.

磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PTS）介导浓度使用独特的机制在细菌中摄取糖的糖，并使用磷酸化糖基材与运输协同以驱动浓度梯度的吸收。 PTS 包括几种可溶性磷酸转移酶和一个整体膜蛋白EIIC。 EIIC组件负责底物特异性，整个膜易位，对于磷酸化反应进行。我们最近在一个X射线结构中解决了EIIC同源物CHBC的X射线结构向内倾斜的构象，这使我们能够为构象变化提出假设这将底物跨过双层以及如何发生磷酸化反应。使用唯一的 MPSDC成员实验室和核心设施提供的资源和专业知识，我们打算使用多种结构，光谱和计算技术来检验这些假设并构建一个 EIIC运输周期的结构动力学的综合模型。具体来说，我们将结合X射线晶体学，鹿EPR，SMFRET和分子动力学模拟，以确定运输蛋白的向外开放和向内开放的构象，并确定eiic如何形成三元及其糖基质和可溶性PTS组件EIIB的复合物，以催化磷酸化反应。