INTERMEDIATES AND PROTEIN FOLDING

本项目的目标是了解蛋白质折叠中动力学障碍的性质，并确定部分结构化中间体在该反应中所起的作用。 这些经常被观察到的物种是目前划分蛋白质如何折叠的假设的争议的中心。 一种观点认为折叠是一个过程，通过离散的中间体在一个明确的途径进行。 另一种认为折叠是状态和路径的连续体，中间体是能量景观中的可选坑。 为了验证这些假设，已经开发了一种新的方法，用于根据蛋白质折叠的速度来区分蛋白质序列。 这种动力学选择将用于从脱辅基肌红蛋白突变体的组合文库中分离快速和慢速折叠变体。 导致快速和缓慢折叠的突变的类型、位置和结构背景将确定脱辅基肌红蛋白折叠中的限速相互作用。 将构建单个快速和缓慢折叠突变体。 停流荧光实验将阐明其动力学折叠机制。 折叠过程中骨架氢键的变化将通过脉冲酰胺氢交换研究来表征。 突变蛋白质在其天然和中间状态的结构将通过核磁共振实验进行研究。 这些联合研究将通过确定突变体的快速和慢速折叠是否具有共同的折叠机制，或者它们是否通过完全不同的途径折叠来测试这两种假设。这项建议提出了处理下列相互关联问题的新办法。(i)中间体会加快折叠速度还是减慢折叠速度？(ii)限速步骤的性质是什么？(iii)动力学障碍是可选的还是内在的？(iv)折叠速度的顺序决定因素是什么？(v)我们能把快速折叠的特性设计成蛋白质吗？ 通过将机械实验与现代组合工具相结合，我们希望界定折叠中间体的结构和性质以及连接它们的能量障碍，它们共同定义了蛋白质折叠的机制。

The goals of this project are to understand the nature of kinetic barriers in protein folding, and to determine the roles that partially structured intermediates play in this reaction. These frequently-observed species lie at the center of the controversy that currently divides hypotheses for how proteins fold. One view regards folding as a process that proceeds through discrete intermediates in a well-defined pathway. The other considers folding as a continuum of states and pathways, with intermediates being optional pits in the energy landscape. To test these hypotheses, a novel method has been developed for discriminating protein sequences based on the speeds at which they fold. This this kinetic selection will be used to isolate fast- and slow-folding variants from combinatorial libraries of apomyoglobin mutants. The types, locations, and structural contexts of the mutations that give rise to fast and slow folding will identify the rate-limiting interactions in apomyoglobin folding. Individual fast- and slow-folding mutants will be constructed. Stop-flow fluorescence experiments will elucidate their kinetic folding mechanisms. Changes in backbone hydrogen bonding during folding will be characterized by pulsed amide hydrogen exchange studies. Structures of mutant proteins in their native and intermediate states will be investigated by nuclear magnetic resonance experiments. These combined studies will test the two hypotheses by determining if fast- and slow-folding of mutants share a common folding mechanism, or if they fold by altogether different routes. This proposal develops new approaches that address the following interrelated questions. (i) Do intermediates speed up or slow down folding? (ii) What is the nature of the rate-limiting step? (iii) Are kinetic barriers optional or intrinsic? (iv) What are the sequence determinants of folding speed? (v) Can we engineer the property of fast folding into proteins? By combining mechanistic experiments with modern combinatorial tools, we hope to delimit the structures and properties of folding intermediates and the energy barriers that link them, which together define the mechanism of protein folding.