Kinetic studies of protein folding

描述（由申请人提供）：描述折叠机制对人类健康和生物功能具有巨大的影响。体内的折叠错误可能导致超过30%的合成多肽的损失，而错误折叠的构象异构体已涉及大量的人类疾病，包括癌症和淀粉样变性。蛋白质稳定性的丧失是75%的单基因疾病的原因。此外，折叠分子伴侣的普遍存在证明了折叠过程在许多细胞活动中的重要性。围绕蛋白质折叠的广泛的生物过程和疾病强调了对这一普遍过程进行机械研究的重要性。目前的建议将结合实验和模拟，以解决有关蛋白质折叠的三个悬而未决的问题。虽然有一个共识，过渡态（TS）采用类似天然的拓扑结构，他们有时被描述为极化，扩大版本的天然状态，要么包含大量的二级结构或形成在一个一般的崩溃周围的扩散核。这种多样性反映了现实吗？或者，这是探测TS的方法不充分的结果，并且存在一个更连贯的图片来描述TS？甚至更多的不确定性围绕着导致TS的早期步骤-是有一个崩溃发生通过多个，不同的路线代表广泛的漏斗，还是有一个占主导地位的有序途径与H-键结构的顺序建立，因为我们已经提出了泛素？从计算的角度来看，一个模拟折叠过程的算法可以预测路径，从而预测天然结构而不利用同源性吗？在目标1中，我们将测试我们的预测，即70%的天然拓扑结构存在于许多蛋白质的TS中，然后研究70%水平的起源。为了做到这一点，我们将应用我们的？分析方法，以表征所选的一组天然存在和设计的蛋白质。在目标2中，我们将提出一种算法，该算法利用折叠过程来预测路径和结构，而无需求助于同源性，并在此过程中测试各种折叠模型。在目标3中，我们将合理地填充早期和晚期中间体，并使用NMR氢交换和弛豫色散方法对其进行表征。我们将进行“蛋白质尸检”，其中埋葬卢？Glu突变驱动pH依赖性亚全局解折叠以填充晚期中间体。公共卫生相关性：描述折叠机制对人类健康和生物功能有着巨大的影响。拟议的研究将确定蛋白质折叠的基本原则，包括导致限速步骤的早期事件的性质，并将利用这些信息来预测途径和结构，而不诉诸同源性。

DESCRIPTION (provided by applicant): Delineating folding mechanisms has tremendous implications for human health and biological function. Folding errors in vivo may be responsible for the loss of more than 30% of synthesized polypeptides, while misfolded conformers have been implicated in a large number of human diseases, including cancer and amyloidoses. The loss of protein stability is the cause of 75% of the monogenic diseases. In addition, the ubiquitous presence of folding chaperones testifies to the importance of the folding process in many cellular activities. The wide range of biological processes and diseases centered around protein folding emphasizes the importance of mechanistic studies of this universal process. This current proposal will integrate experiment and simulations to address three outstanding questions concerning protein folding. Although there is a consensus that transition states (TSs) adopt a native-like topology, they have been described at times as polarized, expanded versions of the native state, either containing extensive amounts of secondary structure or formed in a general collapse around a diffuse nucleus. Does this diversity reflect reality? Or is it the consequence of inadequate methods to probe the TS, and a more coherent picture exists to describe TSs? Even more uncertainty surrounds the early steps leading up to the TS - is there is a collapse occurring via multiple, diverse routes represented by broad funnel, or is there a dominant ordered pathway with a sequential build-up of H-bonded structure, as we have proposed for ubiquitin? From the computational standpoint, can an algorithm that mimics the folding process predict pathways and, consequently, native structures without utilizing homology? In Aim 1, we will test our prediction that 70% of the native topology is present in the TS of many proteins and then investigate the origin of the 70% level. To do this, we will apply our ?-analysis method to characterize a selected set of naturally occurring and designed proteins. In Aim 2, we will advance an algorithm that utilizes the folding process to predict pathways and structure without resorting to homology, and in the process test various folding models. In Aim 3, we will rationally populate early and late intermediates and characterize them using NMR hydrogen exchange and relaxation dispersion methods. We will perform a "protein autopsy" in which buried Leu?Glu- mutations drive a pH-dependent subglobal unfolding to populate late intermediates. PUBLIC HEALTH RELEVANCE: Delineating folding mechanisms has tremendous implications for human health and biological function. The proposed research will identify the basic principles governing protein folding, including the nature of the early events leading to the rate-limiting step, and will use this information to predict pathways and structure without resorting to homology.