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

非技术解释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，但破译大蛋白质折叠的机制仍然是一个艰巨的挑战。这个项目的目的是通过将尖端的实验方法与强大的计算策略结合起来，探索中等大小蛋白质无肌红蛋白的早期折叠阶段，从而从小蛋白质转移到具有更复杂折叠行为的大蛋白质。一组关于微秒时间尺度上填充的中间状态的结构和动力学性质的丰富实验数据将为验证和改进模拟技术提供基准。这些发现将对我们对蛋白质折叠的理解以及分子模拟准确模拟折叠反应的能力提供关键测试，从而产生前所未有的空间和时间分辨率的结构和机制洞察。该项目为未来的科学家提供了广泛的实验、计算和理论方法的培训机会。学生将从实验小组和理论小组之间的密切合作中受益。罗德博士是美国国家科学基金会发起的蛋白质折叠联盟的成员，该联盟的主要任务是促进科学交流和合作。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。深入了解蛋白质折叠的机制具有超越直接领域的影响。例如，非天然蛋白质状态对于理解蛋白质聚集是至关重要的，这在生物技术和医学中具有重大的实际意义。发展的快速动力学技术不仅有利于蛋白质折叠的研究，而且有助于配体结合和酶反应机理的研究。计算模型的关键测试和改进将有利于计算生物学的其他领域，如结构预测、蛋白质相互作用的建模和功能构象变化。本项目的目标是：(I)通过对apoMb(ApoMb)折叠过程中遇到的状态的动力学网络进行详细的实验和计算分析来阐明原型α-螺旋蛋白质的折叠机制；(Ii)了解对启动折叠、定义链拓扑和指导对天然结构的搜索非常重要的氨基酸序列的关键特征。Fox Chase癌症中心的Heinrich Roder团队将结合超快混合方法、荧光和核磁共振检测的H/D交换标记来阐明具有单一残基分辨率的apoMb的动力学折叠动力学。这一结果将为验证坦普尔大学文森特·沃兹小组将要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，结合马尔可夫状态模型方法，将产生原子分辨结构洞察和实验观测的可检验预测。最近的动力学研究表明，apoMb在酸性条件下(pH 4.2)的折叠是一个在启动后250微秒内完成的多阶段过程。这一时间尺度可以通过计算获得，并使用Folding@Home分布式计算机网络使大规模MD模拟成为现实。突变对实验观察物和模拟状态网络的影响将影响折叠启动和路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、突变和基于核磁共振的氢-氚交换方法与最先进的计算方法相结合，将有可能描述153个残基apoMb折叠的早期阶段，其详细程度以前仅针对小得多的蛋白质。实验观测数据，包括速率常数、突变扰动、荧光性质和NH保护模式，将作为测试和改进计算模型的基准，这反过来将提供具有原子分辨率的结构和机制洞察，并做出将在下一轮实验中测试的预测。这些结果将扩大我们对蛋白质折叠原理的理解，使我们从小的两态文件夹扩展到具有复杂的多态折叠行为的更大的螺旋蛋白质，并解决长期存在的问题，涉及折叠起始和繁殖的序列决定因素，以及蛋白质折叠中间体的结构特征和动力学作用。

Non-technical explanationAlthough 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.An 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. 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.