Mining the Structural Genomics Initiative for Disorder

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The goal ofthe proposed research is to experimentallytest for intrinsic disorder amongthe Structural Genomics Initiativeproteins 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 thereforemake up a fraction ofthe proteins that can be purifiedbut that defy attempts at structure determination. To reach the stated goal,the selected proteins will be screened in ahigh-throughput format for intrinsic disorder by protease digestion. Proteolysis will be monitored by SDSgelelectrophoresis (for quantitative digestion rate analysis includingcomparisons with knownstandards, and forimportant clues about the extent of the disorder) and by peptide mass fingerprinting(for assignment of cleavage sites). The proteolysis results willbe compared to computer predictions ofproteolytic sites and disorder, with the expectation that most of the cut sites willbe in regions of predicted disorder and most resistant sites will be in regions of predicted order. Likewise,proteins willbe screened in a high-throughput format for collapsed (molten globule-like)disorder by urea titration ofthe fluorescence ofbound i-anilino-8-napthalene sulfonate (ANS). Those proteins that show loss ofANSfluorescenceat low urea concentrations will be further tested for ANSprotection oftrypsin digestion to reveal the ANS binding region(s). Aselected subset of the proteins will be characterized by fluorescence quenching using acrylamideas compared to trichloroethanol and by near and far UVcircular 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 compareproteins that have been predicted to be ordered (but found to be disordered by NMR) with proteins predicted to be disordered (andfound to be disorderedby 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 variousadditives.]] These methods provide further discrimination between extended (random coil- like) and collapsed disorder. The spectroscopic data willbe compared with specific models and sequence analysis to test hypotheses regardingstructure-sequence relationships for intrinsically disordered proteins. [[The above experiments will be repeated on proteins that yield 3-D structuresto serve as controls. ]] If successful, this workwillincrease the number ofwell-characterized intrinsically disordered proteins.Of special importance is that, as the functions ofthese proteins become determined, the knowledgebaseof disorder-function relationships will expand.Alsoof importance is that this workwill significantly increase the sequence-function informationobtained from the structural genomics initiative with very little incremental increase over the current investment.