Collaborative Research: Early Stages of Protein Folding Explored by Experimental and Computational Approaches

合作研究：通过实验和计算方法探索蛋白质折叠的早期阶段

• 批准号: 1412378
• 负责人: Heinrich Roder
• 金额: $ 101.54万
• 依托单位: Institute For Cancer Research
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1412378&HistoricalAwards=false
• 关键词: Collaborative; Research; Early; Stages; Protein

Non-technical explanationAn in-depth understanding of the mechanisms of protein folding has implications beyond the immediate field. For example, non-native protein states are critical for understanding protein aggregation, which has major practical implications in biotechnology and medicine. The rapid kinetics techniques to be developed will not only benefit protein folding research, but also studies of ligand binding and enzymatic reaction mechanisms. Critical testing and refinement of computational models will benefit other areas of computational biology, such as structure prediction, modeling of protein interactions and functional conformational changes. Although major progress has been made in recent years in understanding how small proteins fold, deciphering the mechanisms of folding of larger proteins remains a daunting challenge. This project is aimed at moving beyond small proteins to larger ones with more complex folding behavior by joining cutting edge experimental approaches with powerful computational strategies for exploring early stages of folding of apomyoglobin, a medium size protein. A rich set of experimental data on the structural and dynamic properties of intermediate states populated on the microsecond time scale will provide benchmarks for validating and refining simulation techniques. The findings will provide a critical test of our understanding of protein folding and the power of molecular simulation to accurately model the folding reaction, yielding structural and mechanistic insight with unprecedented spatial and temporal resolution. This project offers training opportunities for future scientists in a wide range of experimental, computational and theoretical approaches. Students will benefit from the close collaboration between experimental and theoretical groups. Dr. Roder is a member of the NSF-sponsored Protein Folding Consortium whose main mission is to foster scientific exchange and collaboration. Dr. Voelz is a participating researcher in the Folding@home distributed computing project, a unique platform for scientific outreach that promotes public awareness for the importance of basic research in protein folding.Technical descriptionThe objectives of this project entitled "Collaborative Research: Early Stages of Protein Folding Explored by Experimental and Computational Approaches" are: (i) to elucidate the folding mechanism of a prototypic alpha-helical protein by detailed experimental and computational analysis of the kinetic network of states encountered during folding of apomyoglobin (apoMb); (ii) to understand key features of the amino acid sequence important for initiating folding, defining chain topology and directing the search for the native structure. The group of Heinrich Roder at the Fox Chase Cancer Center will combine ultrafast mixing methods with fluorescence and NMR-detected H/D exchange labeling to elucidate the kinetic folding dynamics of apoMb with single-residue resolution. The results will provide a basis for validating computational models to be developed by the group of Vincent Voelz at Temple University. Modeling of apoMb folding dynamics by molecular dynamics (MD) simulation, combined with Markov State Model approaches, will yield atomic-resolution structural insight and testable predictions of experimental observables. Recent kinetic studies have shown that folding of apoMb under acidic conditions (pH 4.2) is a multi-stage process completed within 250 microseconds of initiation. This time scale is computationally accessible and makes large-scale MD simulations a realistic proposition, using the Folding@home distributed computer network. Effects of mutations on experimental observables and the simulated network of states will inform on the sequence determinants for folding initiation and pathway selection.By combining advanced experimental techniques, including kinetic analysis with microsecond resolution, mutagenesis and NMR-based hydrogen-deuterium exchange methods, with state ofthe-art computational methods, it will be possible to describe early stages of folding of the 153 residues apoMb with a level of detail that has previously been achieved only for much smaller proteins. Experimental observables, including rate constants, mutational perturbations, fluorescence properties and NH protection patterns, will serve as benchmarks for testing and refining computational models, which in turn will provide structural and mechanistic insight with atomic resolution and make predictions to be tested in a next round of experiments. The results will extend our understanding of the principles of protein folding beyond small two-state folders to a larger helical protein with complex multi-state folding behavior and address long-standing questions concerning the sequence determinants for folding initiation and propagation, and the structural features and kinetic roles of protein folding intermediates.

非技术解释对蛋白质折叠机制的深入理解具有超出当前领域的意义。例如，非天然蛋白质状态对于理解蛋白质聚集至关重要，这在生物技术和医学中具有重大的实际意义。待开发的快速动力学技术不仅有利于蛋白质折叠研究，还有利于配体结合和酶反应机制的研究。计算模型的关键测试和完善将有利于计算生物学的其他领域，例如结构预测、蛋白质相互作用建模和功能构象变化。尽管近年来在理解小蛋白质如何折叠方面取得了重大进展，但破译较大蛋白质的折叠机制仍然是一项艰巨的挑战。该项目旨在通过将尖端实验方法与强大的计算策略相结合，从小型蛋白质转向具有更复杂折叠行为的较大蛋白质，以探索脱辅基肌红蛋白（一种中等大小的蛋白质）折叠的早期阶段。关于微秒时间尺度上的中间态的结构和动态特性的丰富实验数据将为验证和完善模拟技术提供基准。这些发现将为我们对蛋白质折叠的理解以及分子模拟精确模拟折叠反应的能力提供关键测试，从而以前所未有的空间和时间分辨率产生结构和机制见解。该项目为未来的科学家提供广泛的实验、计算和理论方法的培训机会。学生将受益于实验小组和理论小组之间的密切合作。 Roder 博士是 NSF 资助的蛋白质折叠联盟的成员，其主要任务是促进科学交流与合作。 Voelz 博士是 Folding@home 分布式计算项目的参与研究员，该项目是一个独特的科学推广平台，旨在提高公众对蛋白质折叠基础研究重要性的认识。 技术说明 该项目题为“协作研究：通过实验和计算方法探索蛋白质折叠的早期阶段”，其目标是：(i) 通过以下方式阐明原型 α 螺旋蛋白质的折叠机制： 对脱辅基肌红蛋白 (apoMb) 折叠过程中遇到的状态动力学网络进行详细的实验和计算分析； (ii) 了解对于启动折叠、定义链拓扑和指导寻找天然结构很重要的氨基酸序列的关键特征。 Fox Chase 癌症中心的 Heinrich Roder 团队将超快混合方法与荧光和 NMR 检测的 H/D 交换标记相结合，以单残基分辨率阐明 apoMb 的折叠动力学。结果将为验证天普大学 Vincent Voelz 小组开发的计算模型提供基础。通过分子动力学 (MD) 模拟对 apoMb 折叠动力学进行建模，并结合马尔可夫状态模型方法，将产生原子分辨率的结构洞察力和实验可观测值的可测试预测。最近的动力学研究表明，apoMb 在酸性条件（pH 4.2）下的折叠是一个多阶段过程，在启动后 250 微秒内完成。这个时间尺度是可计算的，并且使用 Folding@home 分布式计算机网络使大规模 MD 模拟成为现实。突变对实验可观测值和模拟状态网络的影响将影响折叠起始和途径选择的序列决定因素。通过将先进的实验技术（包括微秒分辨率的动力学分析、诱变和基于 NMR 的氢-氘交换方法）与最先进的计算方法相结合，将有可能以以前所没有的详细程度描述 153 个残基 apoMb 折叠的早期阶段。 仅针对更小的蛋白质才能实现。实验观测值，包括速率常数、突变扰动、荧光特性和 NH 保护模式，将作为测试和完善计算模型的基准，进而提供具有原子分辨率的结构和机制见解，并做出在下一轮实验中进行测试的预测。这些结果将扩展我们对蛋白质折叠原理的理解，从小型两态折叠扩展到具有复杂多态折叠行为的较大螺旋蛋白质，并解决有关折叠起始和传播的序列决定因素以及蛋白质折叠中间体的结构特征和动力学作用的长期存在的问题。