Mining the Structural Genomics Initiative for Disorder

挖掘无序结构基因组学计划

• 批准号: 7092310
• 负责人: ALAN KEITH DUNKER
• 金额: $ 25.82万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092310
• 关键词: SDS polyacrylamide gel electrophoresis; biotechnology; chemical fingerprinting; circular dichroism; conformation; functional /structural genomics; high throughput technology; mass spectrometry; methylamines; myoglobin; nuclear magnetic resonance spectroscopy; polyethylene glycols; protein folding; protein purification; protein sequence; protein structure function; proteolysis; trypsin

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.

描述（由申请人提供）：拟议研究的目的是在结构基因组学蛋白中实验测试固有障碍，这些蛋白已成功表达和纯化，但（至少到目前为止）失败了，以产生3D结构。要检验的假设是固有无序的蛋白很常见，因此占可以纯化的蛋白质的一部分，但它不必尝试进行结构测定。为了达到既定目标，将通过蛋白酶消化以高通量格式以高通量格式筛选所选蛋白质。蛋白水解将通过SDS凝胶电泳（用于定量​​的消化率分析，包括与已知标准的比较，以及有关该疾病程度的重要线索）和肽质量指纹印刷（用于分配切割位点）。蛋白水解结果将与蛋白水解位点和疾病的计算机预测进行比较，并期望大多数切割部位将位于预测障碍的区域，并且大多数抗性位点将处于预测顺序的区域。同样，将以高通量格式筛选蛋白质，以通过尿素滴定结合的i- anilino-8-萘苯二甲酸盐的荧光滴定（熔融大球状）疾病。 （ans）。那些显示出低尿素浓度下ANS荧光损失的蛋白质将进一步测试，以保护胰蛋白酶消化以揭示ANS结合区域。与三氯乙醇相比，使用丙烯酰胺的荧光淬灭的特征将以各种水平的三氟乙醇和三甲基胺-N-氧化物[[（（（通过ficoll and折叠）以及ficoll and ficoll and ficoll and gyletheylene glycol（to Crownding））。我们将特别比较预测将被预测（但被NMR无序）的蛋白质与预测为无序的蛋白质（发现并被NMR无序）进行比较。在这里，我们正在测试这样的想法：某些有序的蛋白质由于条件不适当而无法形成结构，但是可以通过这些各种添加剂引起这种蛋白质形成结构。]]这些方法提供了在扩展（随机线圈样）和崩溃的疾病中进一步歧视。光谱数据将与特定模型和序列分析进行比较，以测试有关本质上无序蛋白的结构序列关系的假设。 [[上述实验将在产生3-D结构以作为对照的蛋白质上重复。特别重要的是，随着这些蛋白质的功能的确定，障碍功能关系的知识基础将会扩大。同样重要的是，这项工作将大大增加从结构基因组学计划获得的序列功能信息，而与当前投资相比，增量较小。