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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们正在继续研究CLC型氯化物转运蛋白中离子转运的详细机制。这项工作涉及检查功能丰富的突变蛋白的晶体结构。我预测，我们现在要回答的两个基本问题 - 这将使我们在下一个周期中占领我们 - （1）质子通过该传输蛋白的运动以及（2）Cl-/H+交换途径的性质。 蛋白质CLC-EC1是来自E大肠杆菌的Cl-/H+交换器。它通过未知的传输机制催化了H+的Cl-的电学，化学计量学的trasnmmbrane交换。该蛋白质显示出位于大致跨膜方向的3个Cl离子的结合位点，因此我们对阴离子途径有一个很好的了解。但是，尚不清楚Cl沿该途径的运动如何沿相反方向耦合到H+传输。 由于过去一年的结构和功能工作，其中一些在NSLS中，我们已经确定了将质子从水溶液中转染到蛋白质内部所需的两个关键的谷氨酸重塑 - 一种朝向蛋白质内部 - 一个面向细胞外溶液，另一个面向细胞内溶液。这两个残基被大约20埃分离，我们现在正在寻找质子在质子通过该蛋白质跨膜时“跳”的质子位点。 为了在这项研究中具有机械意义，我们需要三种基本的信息类型：（1）突变体的电生理行为，（2）突变体的晶体结构，以及（3）哈利德结合位点的位置和占用，通过由br ions替代的cl-替代产生的哈利德结合位点的位置和占用。 Prtooin与Fab Fragment结合结晶，并衍射为3.0-3.5 A分辨率。我们正在努力改善这一点，但足以攻击我们目前正在解决的问题。