Folding Mechanisms of Dimeric Beta-Barrel Proteins

描述(申请人提供)：这项提案的总体目标是阐明一对二聚体β-桶蛋白，非人类超氧化物歧化酶和H1V-1蛋白折叠并组装成其天然的功能形式的机制。连续流动、停止流动和手动混合方法的组合，与光学探测相连接，将提供在微秒到千秒时间范围内的折叠事件。动力学和平衡数据将与动力学和热力学模型进行全局匹配，从而能够提取反应中各个步骤的微观速率常数。通过小角x射线散射、荧光各向异性和Forster共振能量转移技术，可以获得有关过渡中间体的大小、形状和结构的信息。利用自然态氢交换法和急冷流氢交换法，结合质谱学，将监测稀有的部分折叠状态在平衡条件下的二级结构和折叠过程中二级结构的发展。从光学和氢交换研究中获得的结构信息将与GO类和分子动力学模拟的预测进行比较。在β链、环和亚基界面的选定位置进行定点突变，将测试单个侧链对这些二聚体蛋白质的折叠、结合和稳定性的作用。对结构/机理关系的洞察将加强从指导折叠自由能表面上的反应的序列和/或子结构中提取共性。Apo超氧化物歧化酶自然突变对折叠的热力学和动力学性质的影响可能为肌萎缩侧索硬化症的分子基础提供有用的见解。更广泛地说，这项研究的结果有望提供多亚单位复合体组装的模型和范例。

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.