INTERMEDIATES AND PROTEIN FOLDING

该项目的目标是了解蛋白质折叠中动力学屏障的性质，并确定部分结构的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关于蛋白质如何折叠的各种假说争论的中心。一种观点认为折叠是一个过程，在一个明确的途径中通过离散的中间体进行。另一种认为折叠是状态和路径的连续体，中间产物是能量景观中可选的坑。为了验证这些假设，已经开发出一种基于蛋白质折叠速度来区分蛋白质序列的新方法。这种动态选择将用于从无肌红蛋白突变体的组合文库中分离出快速和缓慢折叠的变体。产生快速和缓慢折叠的突变的类型、位置和结构背景将确定无肌红蛋白折叠中的限速相互作用。个体快速和缓慢折叠突变体将被构建。停止流动荧光实验将阐明它们的动力学折叠机制。折叠过程中主氢键的变化将通过脉冲酰胺氢交换研究来表征。突变蛋白在其天然和中间状态下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折叠和缓慢折叠是否共享一个共同的折叠机制，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。本建议提出了解决下列相关问题的新方法。(i)中间体加速还是减慢折叠速度？（ii）限制利率步骤的性质是什么？（iii）动能障碍是可选的还是固有的？（iv）决定折叠速度的顺序是什么？(v)我们能否设计出快速折叠成蛋白质的特性？通过将机械实验与现代组合工具相结合，我们希望确定折叠中间体的结构和性质以及连接它们的能量势垒，这些结构和性质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.