Pilot 1: Investigating Conformational Changes in Saccharide Transporter

试点 1：研究糖转运蛋白的构象变化

• 批准号: 9351552
• 负责人: Ming Zhou
• 金额: $ 13.64万
• 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 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9351552
• 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; Crystallization; Cysteine; Cytoplasm; Cytosol; Data; Dimerization; Docking; Electrons; Energy Transfer; Enzymes; Face; Family; Fluorescence; Fostering; Free Energy; Glucose Transporter; Goals; Histidine; Homologous Gene; Integral Membrane Protein; Lanthanoid Series Elements; Mediating; Membrane; Membrane Proteins; Metabolic; Methodology; Methods; Molecular; Molecular Conformation; Motion; Mutation; Orthologous Gene; Pathway interactions; Phosphoenolpyruvate; Phosphorylation; Phosphotransferases; Pilot Projects; Process; Proteins; Protomer; Reaction; Resolution; Resources; Roentgen Rays; Sampling; Side; Structural Models; Structural Protein; Structure; Substrate Specificity; System; Techniques; Testing; X-Ray Crystallography; base; conformational conversion; design; dimer; extracellular; inorganic phosphate; member; molecular dynamics; periplasm; prevent; protein transport; simulation; single molecule; sugar; uptake

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) 介导集中 通过独特的机制，利用细菌内膜的磷酸化来摄取糖 糖底物与运输相配合，以逆浓度梯度驱动摄取。 PTS 包含几种可溶性磷酸转移酶和一种整合膜蛋白 EIIC。 EIIC 组件 负责底物特异性、跨膜易位，并且对于 磷酸化反应继续进行。我们最近解决了 EIIC 同系物 ChbC 的 X 射线结构 内闭塞构象，这使我们能够提出构象变化的假设 使底物穿过双层以及磷酸化反应如何发生。利用独特的 借助MPSDC的成员实验室和核心设施提供的资源和专业知识，我们打算利用 各种结构、光谱和计算技术来测试这些假设并建立一个 EIIC 运输循环的结构动力学综合模型。具体来说，我们将结合X射线 晶体学、DEER EPR、smFRET 和分子动力学模拟来确定结构 转运蛋白的向外开放和向内开放构象，并确定 EIIC 如何形成三元体 与其糖底物和可溶性 PTS 成分 EIIB 形成复合物以催化磷酸化 反应。