Mining the Structural Genomics Initiative for Disorder

描述(申请人提供)：这项拟议研究的目标是对结构基因组学倡议蛋白质中的内在紊乱进行实验测试，这些蛋白质已经成功地表达和纯化，但(至少到目前为止)未能产生三维结构。要检验的假设是，本质上无序的蛋白质很常见，因此只占可以提纯但不利于结构确定的蛋白质的一小部分。为了达到规定的目标，选定的蛋白质将通过蛋白酶消化以高通量形式筛选出内在障碍。蛋白质降解将通过十二烷基硫酸钠凝胶电泳法(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)和多肽质量指纹图谱(用于指定切割位点)进行监测。蛋白质分解的结果将与计算机对蛋白质分解位点和无序的预测进行比较，预计大多数切割位点将位于预测无序的区域，而大多数耐药位点将位于预测顺序的区域。同样，通过尿素滴定结合的i-苯胺基-8-萘磺酸盐的荧光，将以高通量形式筛选坍塌(熔融球状)无序的蛋白质 (ANS)。那些在低尿素浓度下显示ANS荧光丧失的蛋白质将进一步测试ANS对胰酶消化的保护作用，以揭示ANS结合区(S)。与三氯乙醇相比，选定的蛋白质子集将通过使用丙烯酰胺的荧光猝灭以及在不同水平的三氟乙醇和三甲胺-N-氧化物[[(诱导折叠)和Ficoll和聚乙二醇(造成拥挤)]的近紫外圆二色谱和远紫外圆二色谱表征。我们将特别比较那些被预测为有序的蛋白质(但被核磁共振发现为无序)和被预测为无序的蛋白质(并被核磁共振发现为无序)。在这里，我们正在测试这样一种想法，即由于不适当的条件，一些有序的蛋白质无法形成结构，但这些不同的添加剂可以诱导这些蛋白质形成结构。]]这些方法提供了扩展(随机线圈状)和折叠无序之间的进一步区分。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3D结构的蛋白质上重复，作为对照。]]如果成功，这项工作将增加具有良好特性的内在无序蛋白质的数量。特别重要的是，随着这些蛋白质功能的确定，无序-功能关系的知识库将会扩大。同样重要的是，这项工作将显着增加从结构基因组学倡议获得的序列功能信息，而不会比目前的投资增加很少。

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.