Kinetic studies of protein folding

描述(由申请人提供)：描绘折叠机制对人类健康和生物功能具有巨大的影响。体内折叠错误可能导致超过30%的合成多肽丢失，而错误折叠的构象与包括癌症和淀粉样变性在内的大量人类疾病有关。蛋白质稳定性的丧失是75%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广泛的生物过程和疾病强调了对这一普遍过程的机制研究的重要性。这项目前的提议将结合实验和模拟来解决关于蛋白质折叠的三个悬而未决的问题。尽管人们一致认为过渡态(TS)采用了类似于自然的拓扑结构，但它们有时被描述为自然状态的极化、扩展版本，要么包含大量的二级结构，要么在扩散核周围形成普遍的崩塌。这种多样性是否反映了现实？或者，这是不充分的方法探测TS的结果，而有一个更连贯的画面来描述TS？更多的不确定性围绕着通向TS的早期步骤--是否存在通过以宽漏斗表示的多条不同路线发生的崩溃，或者是否存在一条主导的有序途径，其氢键结构顺序积累，就像我们为泛素提出的那样？从计算的角度来看，一个模拟折叠过程的算法能不能在不利用同源性的情况下预测路径，从而预测自然结构？在目标1中，我们将检验我们的预测，即70%的自然拓扑存在于许多蛋白质的TS中，然后调查70%水平的来源。为了做到这一点，我们将应用我们的？分析方法来表征一组自然产生和设计的蛋白质。在目标2中，我们将提出一个算法，该算法利用折叠过程来预测路径和结构，而不需要求助于同源，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，并使用核磁共振氢交换和驰豫分散方法对它们进行表征。我们将进行“蛋白质尸检”，其中埋藏的Leu？Glu突变驱动依赖于pH的亚整体展开，以填充晚期中间体。公共卫生相关性：描绘折叠机制对人类健康和生物功能具有巨大影响。这项拟议的研究将确定支配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，而不需要求助于同源。

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.