CLC-EC1 CHLORIDE-TRANSPORT PROTEIN

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。这项工作涉及功能信息突变蛋白质的晶体结构的检查。我们现在要回答的两个基本问题--我预测，这两个问题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+交换途径的性质。 蛋白质CLC-ec 1是来自大肠杆菌的Cl-/H+交换器。它通过一种未知的转运机制催化化学计量的Cl-跨膜交换H+。该蛋白质显示3个Cl离子的结合位点大致位于跨膜方向，因此我们对阴离子途径有了很好的了解。但是，Cl-离子的运动如何沿沿着这条途径耦合到H+运输在相反的方向是未知的。 作为过去一年结构和功能工作的结果，其中一些是在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盐resdiues-一个面向细胞外溶液，一个面向细胞内。这两个残基相隔约20埃，我们现在正在寻找它们之间的质子化位点，质子在穿过蛋白质时沿着它们“跳跃”。 为了使这项研究的机制意义，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，和（3）的位置和占领的卤素结合位点，通过异常衍射所产生的Br-离子取代Cl-。蛋白与Fab片段组合结晶并衍射至3.0-3.5 A分辨率。我们正在努力改进这一点，但它足以解决我们目前正在解决的问题。

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.