Probing Conformational Change in a Cl-/H+ Antiporter of Known Structure

所有生物体都是由细胞组成的，细胞通过一层薄膜与环境隔开。虽然这种膜形成重要的保护屏障，但它也包括对基本细胞组分的重要屏障。为了克服这个问题，细胞膜含有各种各样的蛋白质，每种蛋白质都专门用于运输特定的货物。这些膜蛋白是动态分子机器，其经历构象变化（形状和结构的变化）以在膜的任一侧拾取和释放货物。理解膜蛋白如何工作的一个主要挑战是定义这些构象变化。CLC蛋白是一类膜蛋白，其特异性地在相反方向上运输氯离子和质子（两种小且带相反电荷的离子）。该项目的目标是了解CLC蛋白如何完成这一壮举：具体来说，定义蛋白质以如此精致的特异性拾取和丢弃每个底物离子的构象变化。这将通过在整个CLC蛋白中定位小（19F）探针并使用核磁共振（NMR）检测它们的运动来实现。通过结合这些信息与静态分子分辨率结构，这是从X射线晶体学已知的，CLC蛋白质功能的动态模型将被创建。更广泛的影响在这个研究项目中描述的实验将涉及培养一名研究生和博士后科学家在生物化学和生物物理方法和分析，这将提供在学术界或生物技术部门的职业生涯的良好准备。这项研究还将涉及本科暑期学生的培训（继续包括妇女和少数民族），这将起到双重作用：（1）为年轻的潜在未来科学家提供研究经验，（2）为领导实验的研究生和博士后提供指导经验。该项目的PI将继续招募和指导妇女和少数民族，参加生物物理学会妇女职业机会委员会，并为斯坦福大学的学生制定指导方案。此外，她将扩大她的作用，包括推广到当地学校。

All living organisms are composed of cells that are separated from their environment by a thin membrane. Although this membrane forms an important protective barrier, it also comprises a significant barrier to essential cellular components. To overcome this problem, cell membranes contain a broad assortment of proteins that are each specialized to transport specific cargo. These membrane proteins are dynamic molecular machines that undergo conformational change (change in shape and structure) to pick up and release cargo on either side of the membrane. A major challenge to understanding how membrane proteins work is to define these conformational changes. The CLC proteins are a class of membrane proteins that specifically transport chloride and protons (two small and oppositely-charged ions) in opposite directions. The goal of this project is to understand how the CLC proteins accomplish this feat: specifically, to define the conformational changes by which the protein pick up and drops off each substrate ion with such exquisite specificity. This will be accomplished by positioning small (19F) probes throughout the CLC protein and detecting their motion using nuclear magnetic resonance (NMR). By combining this information with the static molecular-resolution structure that is known from X-ray crystallography, a dynamic model of CLC protein function will be created.Broader ImpactsThe experiments described in this research project will involve training a graduate student and a postdoctoral scientist in biochemical and biophysical methods and analysis that will provide excellent preparation for a career in academia or in the biotechnology sector. This research will also involve the training of undergraduate summer students (with continued inclusion of women and minorities), which will serve a twofold purpose of (1) providing research experiences to young, potential future scientists and (2) providing mentoring experience to the graduate student and post-doc who are leading the experiments. The PI of this project will continue to recruit and mentor women and minorities, to participate in the Biophysical Society's Committee for Professional Opportunities for Women, and to develop mentoring programs for students at Stanford. In addition, she will broaden her role to include outreach to the local schools.