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 will conduct feasibility studies to determine whether it is possible to rapidly interrogate the entire proteome of an organism and determine which expressed genes bind which metals and what the oxidation state and structural environment of these metals are (a technology we call high-throughput x-ray absorption spectroscopy, HT XAS). Mike Adams (University of Georgia) will provide as test samples a selection of purified proteins from expressed open reading frames (ORFs) of Pyrococcus furiosus (Pf), which is a model organism for the Southeast Collaboratory for Structural Genomics. The current NIH-funded structural genomics efforts (including those at SECSG) are focused on high-throughput (HT) x-ray crystallography and/or HT NMR solution structure determination. We submit that HT XAS could provide useful complementary structural information for a subset of a given proteome and that XAS should have a place as a structural genomics technique. 'Metalloproteomics' experiments based on this technology would explore changes in metalloprotein expression patterns or changes in metal loading of proteins, as a function of altered growth conditions. Our current implementation of HT XAS involves HT cloning, expression, and purification of a large subset of the ORFs of the Pf genome, followed by arraying samples of the gene products in a 5x5 set of 3- L wells in a plastic plate that fits within the current LHe cryostat sample space. We use a 2d raster mapping technique, monitoring specific X-ray emission energies, to first define the metal distribution among this 25-ORF sampling of the proteome. This is followed by spectroscopic analysis of specific wells, based on the determined metal distribution. It is this mapping that makes this project a candidate for using the imaging system on BL9-3. Our mapping is essentially a lower-resolution imaging of our sample array.
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