THIOL-DISULFIDE EXCHANGE BETWEEN N-TERMINAL AND C-TERMINAL ACTIVE SITES IN THIOR

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。 硫氧还蛋白还原酶（TR）是硫氧还蛋白系统的必需酶，其主要功能是防止或修复细胞氧化以及维持负责DNA合成和控制某些细胞信号通路的机制。TR是人类中仅有的25种蛋白质之一，其需要微量元素硒作为氨基酸硒代半胱氨酸（Sec）掺入保守的C-末端活性位点四肽基序中。所有哺乳动物TR都有这种需求，但来自低等动物的TR（如黑腹果蝇（DmTR））则没有，后者利用半胱氨酸（Cys）。本项目的目的是通过晶体学研究TR的含Sec和含Cys活性位点的结构组分，因为目前的结构缺乏理解这些蛋白质的机制所需的关键C-末端结构信息。 迄今为止，没有哺乳动物TR结构可视化的C-末端活性位点。 我们有证据表明，包含C-末端活性位点的C-末端尾部的组成和构型在其与黄素相关的二硫醇（N-末端活性位点）的交换中是重要的。 为了研究这一点，我们已经开发了用各种组成的八肽浸泡在其C-末端缺少8个AA的DmTR晶体（DmTR "8）以确定其结构的方法。使用Rapidata 2008获得的数据，能够确定占有率约为40%的肽。 我们已修改我们的浸泡条件和成分，以提高我们的入住率。 我们的几个八肽包括Sec（硒代半胱氨酸），因此异常散射将有助于分配这些残基。

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. Thioredoxin Reductase (TR) is the essential enzyme of the thioredoxin system, which functions primarily to prevent or repair cellular oxidation as well as maintaining the machinery responsible for DNA synthesis and control of certain cell signaling pathways. TR is one of only 25 proteins in humans that require the trace element selenium incorporated as the amino acid selenocysteine (Sec) in a conserved C-terminal active site tetrapeptide motif. This requirement is shared by all mammalian TRs but not TRs from lower animals such as Drosophila melanogaster (DmTR) which utilizes cysteine (Cys). The purpose of this project is to investigate the structural components of the Sec-containing and Cys-containing active sites of TR by crystallography as current structures lack the critical C-terminal structural information required for understanding the mechanism of these proteins. To date no mammalian TR structure has visualized the C-terminal active site. We have evidence that both composition and configuration of the C-terminal tail which contains the C-terminal active site are important in its exchange with the flavin associated dithiol (N-terminal active site). To investigate this we have developed methods for soaking DmTR crystals lacking 8 AA at their C-termini (DmTR¿"8) with octapeptides of various compositions to determine their structure. Using data obtained at Rapidata 2008 have been able to determine peptides with about 40% occupancy. We have modified our soaking conditions and compositions to improve our occupancy. Several of our octapeptides include Sec (selenocysteine) thus anomalous scattering will be helpful in assigning those residues.