EARLY EVENTS IN PROTEIN FOLDING

蛋白质折叠的早期事件

• 批准号: 6519696
• 负责人: RICHARD BRIAN DYER
• 金额: $ 29.85万
• 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/6519696
• 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.

蛋白质将蛋白质折叠为本地生物活性结构的机制是现代结构生物学的主要未解决问题之一，对理性蛋白质设计，蛋白质结构预测和相关疾病状态的实际影响重要。 该建议的核心目的是表征蛋白质折叠中早期事件的分子动力学和机制。从根本上讲，需要进行快速启动和折叠反应表征的新方法，以实现这一目标。 使用激光诱导的温度跳跃和时间分辨的红外光谱法在实验中已经建立了这种方法的生存能力。 因此，首次可以启动和遵循蛋白质折叠的动力学或以50 ps的时间尺度进行的动力学，或比传统（快速混合）动力学研究快107倍。这项工作的组织问题是，在孤立的肽和蛋白质中二级结构形成的动力学和机制是什么？二次结构形成在指导折叠过程和稳定早期中间体中的作用是什么？ 具体而言，将使用激光诱导的，冲动的宏观施法pH的温度来开发快速蛋白质折叠的新方法。 时间分辨的红外光谱法将用作蛋白质折叠中间体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识别静态和时间分辨红外光谱中的特定结构。 将研究问题的层次结构。将从一系列从头肽开始，将研究螺旋，转弯和薄板形成的动力学和机制。 将探索成核和传播的速率，以及局部相互作用（静电，盐桥，末端上限）的作用。 其次，将探索apomyoglobin快速折叠核心碎片折叠的动力和机制。 局部相互作用的影响再次是焦点，尤其是反向转弯和螺旋的形成，以及它们在小肽的折叠反应中的相对作用。 最后，将研究两种蛋白质蛋白质（球形螺旋蛋白）和CSPA（快速折叠β-桶）的折叠动力学。 这项工作的重点是确定局部和非本地和非本地（内部）相互作用在塑造能量景观中的作用，从而使蛋白质折叠的动力学。