Early Events in Protein Folding

描述（由申请人提供）：本提案的中心目标是描述蛋白质折叠早期事件的动力学和分子机制。我们试图确定基本过程的特征速率和潜在的分子机制，包括二级结构的形成，疏水崩溃和特定三级相互作用的形成。我们计划解决的基本问题包括：关于折叠的能量格局，动力学告诉了我们什么？局域和非局域相互作用在形成能量格局和折叠动力学中的作用是什么？在折叠过程中，早期事件如何影响后续事件？除了动力学对理解蛋白质折叠机制的根本重要性之外，蛋白质快速折叠是否具有内在的生物学意义？从根本上说，需要新的方法来快速引发和结构特异性表征折叠反应，以实现这一目标。在最后的拨款期内，我们继续开拓这种方法的发展，建立了激光诱导温度跳变和pH跳变与时间分辨光谱方法相结合的可行性。我们强调完善的时间分辨红外（TRIR）和荧光技术作为折叠动力学的结构特异性探针。我们建议在系统层次结构中解决上述问题：小的，从头开始的模型肽（螺旋，β -sheet，匝，卷曲线圈），以确定主要过程的动力学；小螺旋蛋白，包括En_HD、BdpA和apoMb结构域；和一个快速折叠b-sheet蛋白，这是E. coil （CspA）的主要冷休克蛋白。模型肽研究为解释蛋白质研究中的早期事件提供了重要的基础。这些努力隐含着对快速起始方法和时间分辨结构特异性光谱技术的持续改进。我们希望这些结果能够阐明这些系统中折叠的具体细节，以及更一般的折叠原理。将追求以下具体目标：1)确定肽模型中二级结构形成的动力学；2)研究小的单域螺旋蛋白的折叠动力学。3)研究小β -薄片蛋白（CspA）的折叠动力学。

DESCRIPTION (provided by applicant): The central objective of this proposal is to characterize the dynamics and molecular mechanisms of the early events in protein folding. We seek to determine the characteristic rates and underlying molecular mechanisms of the fundamental processes, including secondary structure formation, hydrophobic collapse and formation of specific tertiary interactions. The basic questions we plan to address include: What do the dynamics tell us about the energy landscape of folding? What is the role of local versus nonlocal interactions in shaping the energy landscapes and thus the folding dynamics? How do the early events influence subsequent events in the folding process? Beyond the fundamental importance of the dynamics to the understanding of protein folding mechanisms, is there intrinsic biological significance of rapid protein folding? Fundamentally new approaches to the rapid initiation and structure specific characterization of folding reactions are required to fulfill this objective. We have continued to pioneer the development of such approaches in the last grant period, establishing the viability of laser-induced temperature jump and pH jump coupled with time-resolved spectroscopic methods. We emphasize well-established time-resolved infrared (TRIR) and fluorescence techniques as structure-specific probes of folding dynamics. We propose to address the issues outlined above in a hierarchy of systems: small, de novo model peptides (helix, beta-sheet, turns, coiled-coils) to determine the dynamics of the primary processes; small helical proteins including the engrailed homeodomain (En_HD), the B domain of protein A (BdpA) and apomyoglobin (apoMb); and a fast folding b-sheet protein, the major cold shock protein of E. coil (CspA). The model peptide studies provide an essential foundation for the interpretation of the early events in the protein studies. Implicit in these efforts is the continued improvement of the fast initiation methods and time resolved structurally specific spectroscopic techniques. We expect the results to illuminate both the specific details of folding in these systems, as well as more general principles of folding. The following specific aims will be pursued: l) Determine the dynamics of secondary structure formation in peptide models; 2) Investigate the folding dynamics of small, single domain helical proteins. 3) Investigate the folding dynamics of a small beta-sheet protein, CspA.