EARLY EVENTS IN PROTEIN FOLDING

蛋白质折叠的早期事件

• 批准号: 6386239
• 负责人: RICHARD BRIAN DYER
• 金额: $ 28.98万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386239
• 关键词: acidity /alkalinity; chemical kinetics; conformation; hydrogen bond; infrared spectrometry; intermolecular interaction; lasers; method development; molecular dynamics; myoglobin; physical model; protein denaturation; protein folding; protein sequence; protein structure; stress proteins; structural biology; synthetic peptide; temperature jump; thermodynamics; time resolved data

The mechanism by which a protein folds to its native, biologically active structure is one of the major unsolved problems of modern structural biology, with important practical consequences for rational protein design, protein structure prediction and folding related disease states. The central objective of this proposal is to characterize the molecular dynamics and mechanisms of early events in protein folding. Fundamentally new approaches to the rapid initiation and characterization of folding reactions are required to fulfill this objective. The viability of such an approach has been established in experiments using a laser-induced temperature jump and time-resolved infrared spectroscopy. Thus, for the first time, it is possible to initiate and follow the kinetics of protein folding or unfolding on a time scale of 50 ps,or some 107 times faster than conventional (rapid mixing) kinetics studies. The organizing question of this work is what are the dynamics and mechanisms of secondary structure formation in an isolated peptide and in a protein, and what is the role of secondary structure formation in guiding the folding process and stabilizing early intermediates. Specifically, new methods for the rapid initiation of protein folding will be developed, using a laser-induced, impulsive macroscopic perturbation of temperature of pH. Time-resolved infrared spectroscopy will be used as a structure specific probe of the dynamics of protein folding intermediates. The amide and sidechain vibrations of peptides and proteins, together with isotopic labeling will be used to identify specific structures in static and time-resolved infrared spectra. A hierarchy of problems will be investigated. Starting with a series of de novo peptides, the dynamics and mechanism of helix, turn and sheet formation will be investigated. The rate of nucleation and propagation, and the role of local interactions (electrostatic, salt bridges, end capping) will be explored. Second, the dynamics and mechanism of folding of fragments of the fast folding core of apomyoglobin will be explored. The influence of local interactions is again the focus, particularly the formation of reverse turns and helices, and their relative roles in the folding reactions of small peptides. Finally, the folding dynamics of two proteins, apomyoglobin (globular helical protein) and CspA (fast-folding beta-barrel) will be investigated. A key focus of this work is to establish the role of local and nonlocal (intrachain) interactions in shaping the energy landscape, hence the dynamics of protein folding.

蛋白质折叠成其天然生物活性结构的机制是现代结构生物学的主要未解决的问题之一，对于合理的蛋白质设计、蛋白质结构预测和折叠相关的疾病状态具有重要的实际意义。 该建议的中心目标是表征蛋白质折叠早期事件的分子动力学和机制。从根本上说，新的方法来快速启动和表征折叠反应，需要实现这一目标。 这种方法的可行性已经建立在实验中使用激光诱导的温度跳变和时间分辨红外光谱。 因此，对于第一次，它是可能的启动和遵循的动力学的蛋白质折叠或展开的时间尺度为50 ps，或一些107倍，比传统的（快速混合）动力学研究。这项工作的组织问题是什么是一个孤立的肽和蛋白质中的二级结构形成的动力学和机制，以及二级结构形成在指导折叠过程和稳定早期中间体中的作用。 具体而言，将开发新的方法用于快速启动蛋白质折叠，使用激光诱导的，脉冲宏观扰动的pH值的温度。时间分辨红外光谱将被用作蛋白质折叠中间体的动力学的结构特异性探针。 肽和蛋白质的酰胺和侧链振动以及同位素标记将用于在静态和时间分辨红外光谱中识别特定结构。 将研究一系列问题。从一系列的从头肽，动力学和机制的螺旋，转弯和折叠的形成将进行研究。 将探讨成核和传播的速率，以及局部相互作用（静电，盐桥，封端）的作用。 第二，探讨脱辅基肌红蛋白快速折叠核心片段的折叠动力学和机制。 局部相互作用的影响再次成为焦点，特别是反向转角和螺旋的形成，以及它们在小肽折叠反应中的相对作用。 最后，将研究两种蛋白质的折叠动力学，即脱辅基肌红蛋白（球状螺旋蛋白）和CspA（快速折叠β-桶）。 这项工作的一个关键重点是建立本地和非本地（链内）相互作用在塑造能量景观的作用，因此蛋白质折叠的动力学。