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。 硫氧还蛋白还原酶(TRR)是硫氧还蛋白系统的关键酶，其主要功能是防止或修复细胞氧化，以及维持负责DNA合成和某些细胞信号通路控制的机制。TR是人类仅有的25种蛋白质之一，需要将微量元素硒作为氨基酸硒半胱氨酸(Sec)掺入到保守的C末端活性部位四肽基序中。所有哺乳动物的受体都有这一要求，但低等动物如利用半胱氨酸的黑腹果蝇(Dmtr)的受体则不然。本项目的目的是通过结晶学方法研究含SEC和含Cys的受体活性部位的结构组成，因为目前的结构缺乏理解这些蛋白质机制所需的关键C-末端结构信息。到目前为止，还没有哺乳动物的tr结构显示出C末端的活性部位。我们有证据表明，包含C-末端活性部位的C-末端尾部的组成和构型在其与黄素相关的二硫醇(N-末端活性部位)的交换中都是重要的。为了研究这一点，我们开发了用不同组成的八肽浸泡C-末端缺少8个AA的DMTR晶体(DMTR？“8)的方法来确定它们的结构。使用Rapidata 2008获得的数据已经能够确定约40%占有率的多肽。我们已经改变了浸泡条件和组成以改善我们的占有率。我们的几个八肽包括SEC(硒半胱氨酸)，因此异常散射将有助于指定这些残基。

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.