Pilot 1: Investigating Conformational Changes in Saccharide Transporter

磷酸烯醇式丙酮酸依赖的磷酸转移酶系统(PTS)介导浓缩性 通过一种独特的机制通过细菌内膜摄取糖，使用磷酸化的 糖底物与运输相协调，以推动与浓度梯度相反的吸收。临时秘书处 包括几种可溶性磷酸转移酶和一个完整的膜蛋白EIIC。EIIC组件 负责底物的专一性，跨膜转运，是 磷酸化反应继续进行。我们最近解决了一个EIIC同系物的X射线结构，ChbC，在一个 向内遮挡构象，这使得我们可以提出构象变化的假说 这将使底物穿过双层，并了解磷酸化反应如何发生。使用唯一的 由MPSDC的成员实验室和核心设施提供的资源和专业知识，我们打算使用 各种结构、光谱和计算技术来测试这些假设并建立 EIIC运输循环结构动力学的综合模型。具体地说，我们将结合X射线 结晶学，鹿EPR，smFRET和分子动力学模拟，以确定结构 转运蛋白的外向开放和向内开放构象，并确定EIIC如何形成三元 与其糖底物和可溶性PTS组分EIIB形成复合物，以催化磷酸化 反应。

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.