Mining the Structural Genomics Initiative for Disorder

这项研究的目的是通过实验来测试结构性的内在无序， 已成功表达和纯化但失败（至少 到目前为止）产生3D结构。有待检验的假设是，内在无序的蛋白质是 因此构成了一小部分蛋白质，这些蛋白质可以被纯化， 结构测定为了达到预定的目标，将对所选蛋白进行高通量筛选 通过蛋白酶消化的内在障碍形式。将通过SDS凝胶电泳监测蛋白水解 (for定量消化率分析，包括与已知标准品的比较， 关于疾病程度的线索）和肽质量指纹（用于分配切割位点）。 蛋白水解结果将与计算机预测的蛋白水解位点和紊乱进行比较， 预期大多数切割位点将位于预测的紊乱区域，并且大多数抗性位点将位于 在预测的顺序区域。同样，将以高通量形式筛选蛋白质以寻找塌陷的蛋白质 尿素滴定法测定I-苯胺基-8-萘磺酸盐的类熔融球无序荧光 （ANS）。那些在低尿素浓度下显示ANS荧光丧失的蛋白质将被进一步检测 用胰蛋白酶消化ANS保护，以显示ANS结合区。一个选定的蛋白质子集 将通过使用丙烯酰胺与三氯乙醇相比的荧光猝灭来表征， 近紫外和远紫外圆二色性在不同水平的三氟乙醇和三甲基胺-N-氧化物[[（至 诱导折叠）以及聚蔗糖和聚乙二醇（引起拥挤）。我们将特别比较蛋白质 已经被预测为有序的（但通过NMR发现是无序的）， 无序的（并通过NMR发现无序）。我们在这里测试一些有序蛋白质 由于不适当的条件而不能形成结构，但这些蛋白质可以被诱导形成结构 这些不同的添加剂]]这些方法提供了延伸的（无规卷曲）和延伸的（无规卷曲）之间的进一步区分。 像）和崩溃的无序。光谱数据将与特定的模型和序列进行比较 分析以检验关于内在无序蛋白质的结构-序列关系的假设。 [[The上述实验将在产生三维结构的蛋白质上重复进行，作为对照。]]如果 如果成功的话，这项工作将增加特征良好的内在无序蛋白质的数量。 特别重要的是，随着这些蛋白质的功能被确定， 功能障碍的关系将扩大。同样重要的是，这项工作将显着增加 从结构基因组学计划中获得的序列功能信息， 比目前投资额增加。

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.