Mining the Structural Genomics Initiative for Disorder

这项拟议研究的目标是通过实验测试结构中的内在紊乱 基因组学倡议已成功表达和纯化但失败的蛋白质(至少 到目前为止)来产生3-D结构。需要检验的假设是，本质上无序的蛋白质是 共同的，因此构成了一小部分可以提纯但不愿尝试的蛋白质 结构确定。为了达到规定的目标，选定的蛋白质将进行高通量筛选 通过蛋白水解酶消化的固有疾病的格式。蛋白质降解将通过SDS凝胶电泳法进行监测 (用于定量消化率分析，包括与已知标准的比较，以及重要的 关于疾病程度的线索)和多肽质量指纹图谱(用于指定切割位点)。 蛋白质分解的结果将与计算机对蛋白质分解位置和无序的预测进行比较， 预计大多数切割部位将位于预测紊乱的区域，而最具耐药性的部位将 在预测顺序的区域中。同样，蛋白质将以高通量的形式进行筛选，以发现崩溃的情况 尿素滴定结合的I-苯胺基-8-萘磺酸盐的(熔融球状)无序 (ANS)。那些在低尿素浓度下表现出ANS荧光丧失的蛋白质将被进一步测试 用于胰酶消化的ANS保护以揭示ANS结合区(S)。选定的蛋白质子集 将通过使用丙烯酰胺而不是三氯乙醇的荧光猝灭来表征，并通过 三氟乙醇和三甲胺-N-氧化物不同水平的近紫外圆二色谱和远紫外圆二色谱[[(to 诱导折叠)和Ficoll和聚乙二醇(造成拥挤)。我们将特别比较蛋白质 已经被预测为有序的(但核磁共振发现是无序的)，蛋白质被预测为 无序(核磁共振发现无序)。我们正在测试这样一种想法，即一些有序蛋白质 由于不适当的条件而不能形成结构，但这种蛋白质可以被诱导形成结构 通过这些不同的添加剂。]]这些方法进一步区分了扩展(随机线圈- 就像)和崩溃的混乱。光谱数据将与特定型号和顺序进行比较 分析以检验关于内在无序蛋白质的结构-序列关系的假设。 [[上述实验将在产生3-D结构的蛋白质上重复作为对照。]]]如果 如果成功，这项工作将增加具有良好特征的内在无序蛋白质的数量。 特别重要的是，当这些蛋白质的功能被确定时， 无序-功能关系将扩大。同样重要的是，这项工作将显著增加 从结构基因组学倡议中获得的序列功能信息非常少 在当前投资的基础上增加增量。

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.