IDEN OF ANION PERMEATION PATHEWAY/BINDING SITES IN GLUTAMATE TRANSPORTERS

这个子项目是许多研究子项目中的一个 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， 因此可以在其他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。 谷氨酸转运蛋白是一种膜蛋白，在神经传递过程中负责谷氨酸从突触间隙中的清除。 谷氨酸转运蛋白是二级转运蛋白，将底物摄取与钠、钾和质子的膜梯度偶联。 除了双偶联离子通量，它们还表现出非偶联阴离子通量，这是门控的基板结合。 最近，我们已经确定了来自Pyroccocus horikoshii的谷氨酸转运蛋白同系物（GltPh）的晶体结构（Nature 431：811-18，2004; Nature 445：387-93，2007）。 因此，已经证明，这种蛋白质捕获其哺乳动物同源物的许多功能特征，包括阴离子渗透。 我们计划使用含有重原子的阴离子，如Br-，SeCN-和AuCN-，试图定位蛋白质中的阴离子渗透途径/结合位点。GltPh晶体通常衍射至4-5分辨率，少数衍射至3.2-3.5。 我们正在制备浸泡在溶液中的晶体，这些溶液中的阴离子被上述化合物取代。 Br-和AuCN-以前曾被我们用于浸泡，不会显著影响晶体的质量。 然而，在过去，我们从未收集过关于这些浸泡的严格数据，这是我们在拟议的实验中想要填补的差距。 对于每次浸泡，我们计划在SeCN-和Br-浸泡的K边缘以及AuCN-浸泡的L-III边缘收集能量扫描和完整数据集。 这项工作还将包括筛选晶体，以获得每种条件下更高的衍射质量。 由于膜晶体每周都有反应，我们选择直接在同步加速器上而不是在内部衍射仪上进行筛选。

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. Glutamate transporters are integral membrane proteins responsible for clearance of glutamate from the synaptic cleft following rounds of neurotransmission. Glutamate transporters are secondary transporters and couple substrate uptake to membrane gradients of sodium, potassium and protons. In addition to thermodynamically coupled ion fluxes they also demonstrate an uncoupled anion flux, which is gated by substrate binding. Recently we have determined the crystal structure of a glutamate transporter homologue from Pyroccocus horikoshii (GltPh) (Nature 431: 811-18, 2004; Nature 445: 387-93, 2007). It has been consequently demonstrated that this protein captures many functional features of its mammalian homologues, including anion permeation. We are planning to use anions containing heavy atoms such as Br-, SeCN- and AuCN- to attempt to locate anion permeation pathway/binding sites in the protein. GltPh crystals diffract routinely to 4-5 ¿ resolution with a small number diffracting to 3.2-3.5 ¿ . We are preparing crystals soaked in solutions that have anions replaced with the above compounds. Br- and AuCN- have been previously used by us for soaks and do not significantly affect quality of the crystals. However, in the past we have never collected rigorous data on these soaks and this is the gap we would like to fill during the proposed experiment. For each soak we plan to collect an energy scan and a full dataset at K edge for SeCN- and Br- soaks and at L-III edge for AuCN-. The work will also include screening crystals for higher diffraction quality for each condition. Because membrane crystals diffract weekly, we opt for screening directly at the synchrotron rather than at the in-house diffractometer.