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，但破译大蛋白质的折叠机制仍然是一个艰巨的挑战。该项目旨在通过结合尖端的实验方法和强大的计算策略来探索无肌红蛋白（一种中等大小的蛋白质）折叠的早期阶段，从小蛋白质转向具有更复杂折叠行为的大蛋白质。在微秒时间尺度上填充的关于中间状态的结构和动态特性的丰富实验数据集将为验证和改进模拟技术提供基准。这一发现将为我们对蛋白质折叠的理解和分子模拟的能力提供一个关键的测试，以准确地模拟折叠反应，以前所未有的空间和时间分辨率提供结构和机制的洞察力。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。学生将受益于实验和理论小组之间的密切合作。Roder博士是nsf赞助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。对蛋白质折叠机制的深入了解具有超越直接领域的意义。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要的实际意义。快速动力学技术的发展不仅有利于蛋白质折叠的研究，也有利于配体结合和酶促反应机理的研究。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、蛋白质相互作用建模和功能构象变化。该项目名为“合作研究：用实验和计算方法探索蛋白质折叠的早期阶段”，其目标是：(i)通过对无肌红蛋白（apoMb）折叠过程中遇到的动力学网络的详细实验和计算分析，阐明原型α -螺旋蛋白的折叠机制；（ii）了解氨基酸序列的关键特征，这些特征对于启动折叠、定义链拓扑结构和指导寻找天然结构至关重要。Fox Chase癌症中心的Heinrich Roder团队将结合超快混合方法与荧光和核磁共振检测的H/D交换标记，以单残基分辨率阐明apoMb的动力学折叠动力学。这些结果将为验证由天普大学的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，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可测试的实验观测预测。最近的动力学研究表明，在酸性条件下（pH 4.2）， apoMb的折叠是一个多阶段的过程，在250微秒内完成。这个时间尺度在计算上是可访问的，并且使用Folding@home分布式计算机网络使大规模MD模拟成为一个现实的命题。突变对实验观察值和模拟状态网络的影响将为折叠起始和途径选择的序列决定因素提供信息。通过结合先进的实验技术，包括微秒分辨率的动力学分析、诱变和基于核磁共振的氢-氘交换方法，以及最先进的计算方法，将有可能描述153个残基apoMb折叠的早期阶段，其细节水平以前只在更小的蛋白质上实现。实验观察，包括速率常数，突变扰动，荧光特性和NH保护模式，将作为测试和改进计算模型的基准，这反过来将提供原子分辨率的结构和机制洞察力，并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，从小的两态文件夹扩展到具有复杂多态折叠行为的更大的螺旋蛋白，并解决有关折叠起始和繁殖的序列决定因素，蛋白质折叠中间体的结构特征和动力学作用等长期存在的问题。

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.