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: 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
• 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

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.

磷酸烯醇化酶依赖的磷酸转移酶系统（PTS）介导了浓度依赖性的细胞内蛋白质的转移。 通过一种独特的机制，利用磷酸化的糖在细菌的内膜吸收。 糖底物与运输协调一致，以驱动逆浓度梯度的摄取。临时秘书处 包括几种可溶性磷酸转移酶和整合的膜蛋白EIIC。EIIC组件 负责底物特异性，跨膜转运，并且是细胞增殖所必需的。 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， 内封闭构象，这使我们能够提出假设的构象变化 使底物穿过双层以及磷酸化反应如何发生。使用所述唯一 利用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 各种结构，光谱和计算技术来测试这些假设，并建立一个 EIIC运输周期结构动力学的综合模型。具体来说，我们将联合收割机 晶体学，DEER EPR，smFRET和分子动力学模拟，以确定的结构， 外向开放和内向开放构象的转运蛋白，并确定如何EIIC形成三元 与其糖底物和可溶性PTS组分EIIB复合以催化磷酸化 反应