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意义。体内折叠错误可能导致超过30%的合成多肽损失，而错误折叠的构象与许多人类疾病有关，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。以蛋白质折叠为中心的各种生物过程和疾病强调了对这一普遍过程进行机制研究的重要性。目前的建议将结合实验和模拟来解决有关蛋白质折叠的三个突出问题。虽然人们一致认为过渡态（TSs）采用与原生态类似的拓扑结构，但它们有时被描述为原生态的极化、扩展版本，要么包含大量的二级结构，要么在弥漫核周围的普遍坍塌中形成。这种多样性是否反映了现实？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像来描述TS ？甚至更多的不确定性围绕着导致TS的早期步骤-是否存在通过以宽漏斗为代表的多种不同途径发生的崩溃，或者是否存在一个主导的有序途径与h键结构的顺序构建，正如我们对泛素提出的那样？从计算的角度来看，模拟折叠过程的算法能否在不利用同源性的情况下预测路径和天然结构？在Aim 1中，我们将测试我们的预测，即70%的天然拓扑存在于许多蛋白质的TS中，然后研究70%水平的起源。为此，我们将应用？-分析一组选定的自然产生和设计的蛋白质的方法。在Aim 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，而不依赖于同源性，并在此过程中测试各种折叠模型。在Aim 3中，我们将合理填充早期和晚期中间体，并使用核磁共振氢交换和弛豫分散方法对它们进行表征。我们将进行“蛋白质解剖”，其中埋藏的Leu？Glu突变驱动ph依赖的亚全球展开以填充晚期中间体。公共卫生相关性：描述折叠机制对人类健康和生物功能具有巨大意义。拟议的研究将确定控制蛋白质折叠的基本原理，包括导致限速步骤的早期事件的性质，并将使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.