The goal of the proposed research is to experimentally test for intrinsic disorder among the Structural Genomics Initiative proteins that have been successfully expressed and purified but that have failed (at least so far) to yield 3-D structures. The hypothesis to be tested is that intrinsically disordered proteins are common and therefore make up a fraction of the proteins that can be purified but that defy attempts at structure determination. To reach the stated goal, the selected proteins will be screened in a high-throughput format for intrinsic disorder by protease digestion. Proteolysis will be monitored by SDS gel electrophoresis (for quantitative digestion rate analysis including comparisons with known standards, and for important clues about the extent of the disorder) and by peptide mass fingerprinting (for assignment of cleavage sites). The proteolysis results will be compared to computer predictions of proteolytic sites and disorder, with the expectation that most of the cut sites will be in regions of predicted disorder and most resistant sites will be in regions of predicted order. Likewise, proteins will be screened in a high-throughput format for collapsed (molten globule-like) disorder by urea titration of the fluorescence of bound i-anilino-8-napthalene sulfonate ? (ANS). Those proteins that show loss of ANS fluorescence at low urea concentrations will be further tested for ANS protection of trypsin digestion to reveal the ANS binding region(s). A selected subset of the proteins will be characterized by fluorescence quenching using acrylamide as compared to trichloroethanol and by near and far UV circular dichroism at various levels of trifluoroethanol and trimethylamine-N-oxide [[(to induce folding) and by ficoll and polyethylene glycol (to cause crowding). We will especially compare proteins that have been predicted to be ordered (but found to be disordered by NMR) with proteins predicted to be disordered (and found to be disordered by NMR). Here we are testing the idea that some ordered proteins fail to form structure due to inappropriate conditions but that such proteins can be induced to form structure by these various additives.]] These methods provide further discrimination between extended (random coil-like) and collapsed disorder. The spectroscopic data will be compared with specific models and sequence analysis to test hypotheses regarding structure-sequence relationships for intrinsically disordered proteins. [[The above experiments will be repeated on proteins that yield 3-D structures to serve as controls.]] If successful, this work will increase the number of well-characterized intrinsically disordered proteins. Of special importance is that, as the functions of these proteins become determined, the knowledge base of disorder-function relationships will expand. Also of importance is that this work will significantly increase the sequence-function information obtained from the structural genomics initiative with very little incremental increase over the current investment. ? ? ?
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