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.
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