CLC-EC1 CHLORIDE-TRANSPORT PROTEIN

CLC-EC1 氯离子转运蛋白

• 批准号: 7726256
• 负责人: Christopher Miller
• 金额: $ 0.55万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-18 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726256
• 关键词: Address; Anions; Behavior; Binding Sites; Carrier Proteins; Chloride Ion; Chlorides; Computer Retrieval of Information on Scientific Projects Database; Coupled; Escherichia coli; Funding; Glutamates; Grant; Humulus; Institution; Ion Transport; Ions; Membrane; Movement; Nature; Pathway interactions; Positioning Attribute; Proteins; Protons; Research; Research Personnel; Resolution; Resources; Site; Solutions; Source; Structure; United States National Institutes of Health; Work; aqueous; extracellular; improved; mutant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are continuing to study the detailed mechanism of ion transport in a CLC-type Chloride transport protein. This work involves the examination of crystal structures of functionally informative mutant proteins. The two basic questions we are now engaged in answering - and that will be occupying us for the next cycle, I predict - concern (1) the movement of protons through this transport protein and (2) the nature of the Cl-/H+ exchange pathways. The protein, CLC-ec1, is a Cl-/H+ exchanger from E coli. It catalyzes electrogenic, stoichiometric trasnmembrane exchange of Cl- for H+ via an unknown transport mechanism. The protein shows binding sites of 3 Cl ions lying in a roughly transmembrane orientation, so we have a pretty good idea of the anion pathway. But how movement of Cl- ions along this pathway is coupled to H+ transport in the opposite direction is unknown. As a result of structure-and-function work of the past year, some of it at NSLS, we have identified two key glutamate resdiues required for tranferring protons from the aqueous solutions into the protein interior - one facing the extracellular solution and one the intracellular. These two residues are separated by about 20 angstroms, and we are now seeking protonatable sites between these that protons "hop" along as they move across the membrane through this protein. In order to make mechanistic sense in this study, we require three basic types of information: (1) electrophysiological behavior of the mutants, (2) crystal structures of the mutants, and (3) positions and occupancies of the halide-binding sites, via anomalous diffraction arising from Br- ions substituted for Cl-. The prtooein crystalizes in combination with a Fab fragement and diffracts to 3.0-3.5 A resolution. We are endeavoring to improve this, but it is adequate for attacking the questions we are currently addressing.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们正在继续研究 CLC 型氯离子转运蛋白中离子转运的详细机制。这项工作涉及检查具有功能信息的突变蛋白的晶体结构。我们现在正在回答的两个基本问题 - 我预测这将在下一个周期占据我们的时间 - 涉及 (1) 质子通过这种转运蛋白的运动和 (2) Cl-/H+ 交换的性质途径。 该蛋白质 CLC-ec1 是来自大肠杆菌的 Cl-/H+ 交换剂。它通过未知的传输机制催化 Cl- 化学计量跨膜交换为 H+。该蛋白质显示 3 Cl 离子的结合位点大致处于跨膜方向，因此我们对阴离子途径有了很好的了解。但 Cl- 离子沿该路径的运动如何与相反方向的 H+ 传输耦合尚不清楚。 作为过去一年的结构和功能工作的结果，其中一些在 NSLS，我们已经确定了将质子从水溶液转移到蛋白质内部所需的两个关键谷氨酸残基 - 一个面向细胞外溶液，一个面向细胞内溶液。这两个残基相距约 20 埃，我们现在正在寻找这些残基之间的可质子化位点，当质子穿过该蛋白质跨膜时，它们会“跳跃”。 为了在本研究中获得机械意义，我们需要三种基本类型的信息：（1）突变体的电生理行为，（2）突变体的晶体结构，以及（3）卤化物结合位点的位置和占用，通过由 Br- 离子取代 Cl- 产生的反常衍射。该蛋白与 Fab 片段结合结晶并衍射至 3.0-3.5 A 分辨率。我们正在努力改进这一点，但这足以解决我们当前正在解决的问题。