Folding Mechanisms of Dimeric Beta-Barrel Proteins

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，载脂蛋白超氧化物歧化酶和H1V-1蛋白酶折叠并组装成其天然功能形式的机制。连续流，停止流和手动混合方法的组合，与光学检测接口，将提供在微到千秒的时间范围内访问折叠事件。动力学和平衡数据将全局拟合动力学和热力学模型，以便提取反应中各个步骤的微观速率常数。利用小角度x射线散射、荧光各向异性和福斯特共振能量转移技术，可以获得瞬态中间体的尺寸、形状和结构信息。将利用原生态和淬火流氢交换法结合质谱法监测稀有、部分折叠态在平衡条件下的二级结构以及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类氧化石墨烯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诱变将测试单个侧链在这些二聚体蛋白的折叠、结合和稳定性方面的作用。对结构/机理关系的深入了解将有助于从引导折叠自由能表面上反应的序列和/或子结构中提取出一般性。自然发生的超氧化物歧化酶突变对折叠热力学和动力学性质的影响可能为肌萎缩性侧索硬化症的分子基础提供有用的见解。更一般地说，本研究的结果有望为多亚基复合物的组装提供模型和范例。

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.