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结构的结构基因组学倡议蛋白质中的内在障碍。要检验的假设是，本质上无序的蛋白质是常见的，因此构成了一部分可以纯化但无法确定结构的蛋白质。为了达到所述目标，将通过蛋白酶消化以高通量形式筛选所选蛋白质的内在紊乱。将通过SDS凝胶电泳（用于定量消化率分析，包括与已知标准品的比较，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）监测蛋白水解。将蛋白水解结果与蛋白水解位点和紊乱的计算机预测进行比较，预期大多数切割位点将在预测紊乱的区域中，并且大多数抗性位点将在预测顺序的区域中。同样地，通过结合的i-苯胺基-8-萘磺酸盐的荧光的尿素滴定，以高通量形式筛选蛋白质的塌陷（熔化的球状）紊乱 （ANS）。将进一步测试在低尿素浓度下显示ANS荧光损失的那些蛋白质的ANS保护胰蛋白酶消化以揭示ANS结合区域。通过使用丙烯酰胺（与三氯乙醇相比）进行荧光猝灭，并通过在不同水平的三氟乙醇和三甲基胺-N-氧化物[[（诱导折叠）和聚蔗糖和聚乙二醇（导致拥挤）]下的近紫外和远紫外圆二色性，对选定的蛋白质子集进行表征。我们将特别比较预测为有序的蛋白质（但通过NMR发现是无序的）和预测为无序的蛋白质（并通过NMR发现是无序的）。在这里，我们正在测试这样一种想法，即一些有序的蛋白质由于不适当的条件而无法形成结构，但这些蛋白质可以被这些不同的添加剂诱导形成结构。这些方法提供了扩展（随机线圈状）和塌陷的疾病之间的进一步区分。光谱数据将与特定的模型和序列分析进行比较，以测试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3D结构的蛋白质上重复，以作为对照。]]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，随着这些蛋白质的功能变得确定，疾病功能关系的知识基础将扩大。同样重要的是，这项工作将显着增加从结构基因组学计划获得的序列功能信息，而目前的投资几乎没有增加。

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.