Folding Mechanisms of Dimeric Beta-Barrel Proteins

二聚体β-桶蛋白的折叠机制

• 批准号: 7410181
• 负责人: C Robert Matthews
• 金额: $ 27.73万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7410181
• 关键词: Amino Acids; Amyotrophic Lateral Sclerosis; Chemicals; Collaborations; Complex; Computer Simulation; Condition; Cuprozinc Superoxide Dismutase; DNA Sequence Rearrangement; Data; Detection; Development; Dimensions; Dimerization; Dissection; Docking; Endopeptidases; Energy Transfer; Equilibrium; Event; Fluorescence; Fluorescence Anisotropy; Free Energy; Goals; Grant; HIV-1 protease; Human; Hydrogen; Individual; Kinetics; Manuals; Mass Spectrum Analysis; Measures; Methods; Microscopic; Modeling; Molecular; Monitor; Mutation; Optics; Peptide Hydrolases; Peptides; Play; Population; Positioning Attribute; Principal Investigator; Property; Proteins; Range; Rate; Reaction; Resolution; Role; Shapes; Side; Site; Site-Directed Mutagenesis; Structure; Superoxide Dismutase; Surface; Techniques; Testing; Thermodynamics; Time; Urea; base; beta barrel; dimer; insight; molecular dynamics; monomer; polypeptide; programs; protein folding; radius bone structure; research study; size

DESCRIPTION (provided by applicant): The overall goal of this proposal is to elucidate the mechanisms by which a pair of dimeric beta-barrel proteins, apo-human superoxide dismutase and H1V-1 protease, fold and assemble into their native, functional forms. A combination of continuous-flow, stopped-flow and manual-mixing methods, interfaced to optical detection, will provide access to folding events over the micro- to kilo-second time range. The kinetic and equilibrium data will be fit globally to kinetic and thermodynamic models to enable the extraction of microscopic rate constants for individual steps in the reaction. Information on the size, shape and structure of the transient intermediates will be obtained with small angle x-ray scattering, fluorescence anisotropy, and Forster resonance energy transfer techniques. Native-state and quench-flow hydrogen exchange methods, combined with mass spectrometry, will be utilized to monitor the secondary structure of rare, partially-folded states under equilibrium conditions and the development of secondary structure during folding. The structural information, available from both the optical and hydrogen exchange studies, will be compared to the predictions of Go-like and molecular dynamics simulations. Site-directed mutagenesis at selected positions in beta strands, loops, and at the subunit interface will test the roles of individual side chains on the folding, association and stability of these dimeric proteins. The insights obtained into the structure/mechanism relationship will enhance the extraction of generalities from the sequences and/or sub-structures that guide the reaction over the folding free energy surface. The effects of naturally-occurring mutations in aposuperoxide dismutase on the thermodynamic and kinetic properties of folding may provide useful insights into the molecular basis of amyotrophic lateral sclerosis. More generally, the results of this study are expected to provide models and paradigms by which multi-subunit complexes assemble.

描述（由申请人提供）：本提案的总体目标是阐明一对二聚体β桶蛋白（脱辅基人超氧化物歧化酶和H1 V-1蛋白酶）折叠并组装成其天然功能形式的机制。连续流、停流和手动混合方法的组合，与光学检测相结合，将提供微秒至千秒时间范围内的折叠事件。动力学和平衡数据将整体拟合动力学和热力学模型，以提取反应中各个步骤的微观速率常数。瞬态中间体的大小，形状和结构的信息将获得与小角X射线散射，荧光各向异性，和福斯特共振能量转移技术。天然状态和猝灭流氢交换方法，结合质谱，将用于监测平衡条件下的稀有，部分折叠状态的二级结构和折叠过程中二级结构的发展。的结构信息，可从光学和氢交换的研究，将进行比较，以类似围棋和分子动力学模拟的预测。在β链、环和亚基界面中的选定位置处的定点诱变将测试单个侧链对这些二聚体蛋白质的折叠、缔合和稳定性的作用。对结构/机制关系的深入了解将增强从序列和/或子结构中提取共性，这些序列和/或子结构引导折叠自由能表面上的反应。脱氧超氧化物歧化酶的自然发生的突变对折叠的热力学和动力学性质的影响可能提供有用的见解肌萎缩侧索硬化症的分子基础。更一般地说，本研究的结果预计将提供多亚基复合物组装的模型和范例。