CLC-EC1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。这项工作涉及到对具有功能信息的突变蛋白质的晶体结构的检查。我们现在正在回答的两个基本问题--我预测，这将在下一个周期中占据我们的位置--涉及(1)质子通过这种运输蛋白的运动和(2)氯-/H+交换途径的性质。 该蛋白CLC-EC1是一种来自大肠杆菌的Cl-/H+交换蛋白。它通过一种未知的转运机制催化氯离子的化学计量跨膜交换。该蛋白显示3个氯离子的结合位置大致位于跨膜方向，因此我们对阴离子途径有一个很好的了解。但氯离子沿这一途径的运动如何与H+在相反方向上的传输相耦合还是个未知数。 作为过去一年结构和功能工作的结果，其中一些工作在NSLS，我们有 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溶液-一个面向细胞外溶液，另一个面向细胞内。这两个残基之间的距离约为20埃，我们现在正在寻找这两个残基之间的质子化位置，当质子穿过膜通过这个蛋白质时，它们会“跳跃”。 为了从机理上理解这项研究，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)通过溴离子取代氯离子产生的异常衍射，卤化物结合部位的位置和占据。原晶体与FAB碎片结合在一起结晶，并衍射到3.0-3.5A分辨率。我们正在努力改进这一点，但这足以解决我们目前正在处理的问题。

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.