Mining the Structural Genomics Initiative for Disorder

描述（由申请人提供）：拟议研究的目标是通过实验测试已经成功表达和纯化但未能（至少到目前为止）产生3-D结构的结构基因组计划蛋白质中的内在紊乱。要验证的假设是，内在无序的蛋白质是常见的，因此构成了可以纯化但无法确定结构的蛋白质的一部分。为了达到既定目标，选定的蛋白质将通过蛋白酶消化以高通量格式筛选内在紊乱。蛋白水解将通过SDS凝胶电泳（用于定量消化率分析，包括与已知标准的比较，以及关于疾病程度的重要线索）和肽质量指纹图谱（用于分配裂解位点）来监测。蛋白质水解结果将与计算机预测的蛋白质水解位点和紊乱进行比较，预计大多数切割位点将位于预测的紊乱区域，而大多数抗性位点将位于预测的有序区域。同样，将通过尿素滴定结合的i-苯胺-8-萘磺酸盐的荧光，以高通量形式筛选蛋白质的崩溃（熔融球状）紊乱

DESCRIPTION (provided by applicant): 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.