Mining the Structural Genomics Initiative for Disorder

挖掘无序结构基因组学计划

• 批准号: 7392642
• 负责人: ALAN KEITH DUNKER
• 金额: $ 27.95万
• 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/7392642
• 关键词: 3-Dimensional; Acrylamide; Acrylamides; Alkanesulfonates; Amino Acids; Behavior Disorders; Binding; Biological; Circular Dichroism; Computers; Condition; Crowding; Data; Databases; Digestion; Discrimination; Disease; Disorder by Site; Endopeptidases; Environment; Exhibits; Failure; Ficoll; Fingerprint; Flowcharts; Fluorescence; Frequencies; Genome; Goals; Helix (Snails); Homology Modeling; In Vitro; Investments; Laboratories; Lead; Ligands; Mass Spectrum Analysis; Methods; Mining; Modeling; Molecular; Molecular Conformation; Monitor; Myoglobin; Nature; Numbers; Oxides; Pattern; Peptide Hydrolases; Peptides; Physiological; Polyethylene Glycols; Polymers; Process; Property; Proteins; Proteolysis; Publishing; Range; Rate; Relative (related person); Reporting; Research; Research Personnel; Resistance; Running; Scanning; Screening procedure; Sequence Analysis; Set protein; Shapes; Site; Staging; Standards of Weights and Measures; Structure; SwissProt; Testing; Titrations; Trifluoroethanol; Trypsin; Urea; Work; Writing; apomyoglobin; clostripain; computer program; cost; dichroism; ear helix; expectation; gel electrophoresis; insight; interest; novel; numb protein; programs; protein function; protein structure; research study; size; structural genomics; three dimensional structure; trimethyloxamine

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.

拟议的研究的目的是在结构上进行内在疾病的实验测试 基因组学启动蛋白已成功表达和纯化但失败了（至少 到目前为止）产生3D结构。要检验的假设是固有无序蛋白是 因此，可以纯化的一小部分蛋白质，因此可以纯化尝试 结构确定。为了达到陈述的目标，选定的蛋白质将以ahigh-thoughtup进行筛选 蛋白酶消化的内在障碍格式。蛋白水解将通过SDSGELELECTROROSIS监测 （用于定量​​消化率分析，包括具有已知标准的综合子，并且很重要 关于该疾病程度的线索）和肽质量指纹（用于分配切割部位）。 蛋白水解结果将与蛋白质水解位点和混乱的计算机预测相比 期望大多数切割站点将在预测的疾病和大多数耐药地点的区域中 在预测秩序的地区。同样，蛋白质将以高通量格式筛选以塌陷 通过尿素滴定I-Anilino-8-硝基磺酸盐的荧光滴定（熔融球状）疾病 （ans）。那些表现出散布低尿素浓度损失的蛋白质将进一步测试 为了使龙骨蛋白消化的Ansprotection揭示ANS结合区域。蛋白质的阿森选择子集 与三氯乙醇相比 在各级三氟乙醇和三甲胺-N-氧化物的近乎近外的紫外二分色素[[[[[ 诱导折叠）和ficoll和聚乙烯乙二醇（引起拥挤）。我们将特别会比较股蛋白 预计将被预测为蛋白质（但被发现被NMR无序）。 无序（NMR无序）。在这里，我们正在测试一些有序蛋白质的想法 由于条件不适当而无法形成结构，但可以诱导这种蛋白质形成结构 ]这些方法通过这些方法提供了进一步的歧视（随机线圈 喜欢）和崩溃的疾病。光谱数据将与特定模型和序列相比 分析以测试假设的固有无序蛋白质的关注结构 - 序列关系。 [[上述实验将在产生3-D结构的蛋白质上重复使用。 ]] 如果 成功的是，这种工作意外是特征的本质上无序蛋白的数量 特别重要的是，随着这些蛋白质的功能的确定，知识库 障碍功能关系的关系将扩大。重要的是，这项工作将大大增加 从结构性基因组学计划中获取的序列功能信息很少 与当前投资相比增长。