Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer

描述（由申请人提供）：对于蛋白质在细胞环境中折叠的方式知之甚少。更具体地说，能够直接评估蛋白质构象的方法在很大程度上尚未探索核糖体结合的新生蛋白质所实现的折叠程度和分子形状。本研究的目的是研究在触发因子 (TF) 伴侣不存在和存在的情况下，核糖体结合的新生蛋白的短程螺旋二级结构和疏水性塌陷（或缺乏）的程度。将检查新生链伸长的不同阶段。将分析源自脱辅基肌红蛋白、apoHmpH 和 Fim H 三种蛋白的新生链。局部二级结构和最重要的疏水性塌陷是体外蛋白质折叠的两个众所周知的主要驱动力。然而，当蛋白质从核糖体中出现时，它们在折叠过程中的重要性尚不清楚。该项目将主要通过福斯特共振能量转移（FRET）进行，通过在受体不存在和存在的情况下测量 FRET 供体的荧光寿命。我们将评估 FRET 效率变化（与分子内距离分布的变化成比例），以监测当新生链从核糖体隧道中出现时核糖体结合模型蛋白的二级和三级结构的变化，以获得有关其局部结构和塌陷程度的证据。鉴于 TF 非极性内表面可以有效地结合新生的不完整蛋白质并显着改变其结构和压缩程度，因此还将研究上述特性对 TF 伴侣存在和不存在的具体依赖性。

DESCRIPTION (provided by applicant): Very little is known about the way proteins fold in the cellular environment. More specifically, the degree of folding and molecular shape achieved by ribosome-bound nascent proteins is largely unexplored by methodologies able to provide a direct assessment of protein conformation. The goal of this research is to investigate the short-range helical secondary structure and degree of hydrophobic collapse (or lack thereof) of ribosome-bound nascent proteins in the absence and presence of the trigger factor (TF) chaperone. Different stages of nascent chain elongation will be examined. Nascent chains derived from the three proteins apomyoglobin, apoHmpH and Fim H will be analyzed. Local secondary structure and, most importantly, hydrophobic collapse are two well known major driving forces for protein folding in vitro. However, nothing is known about their importance in the context of folding as proteins emerge out of the ribosome. This project will be primarily carried out by Forster resonance energy transfer (FRET) via fluorescence lifetime measurement of the FRET donor in the absence and presence of the acceptor. We will assess FRET efficiency variations (proportional to variations in intra-molecular distance distributions) to monitor changes in secondary and tertiary structure of ribosome-bound model proteins as the nascent chains emerge out of the ribosomal tunnel to gain evidence about their degree of local structure and collapse. The specific dependence of the above properties on the presence and absence of the TF chaperone will also be investigated, in light of the fact that the TF nonpolar inner surface may effectively bind the nascent incomplete proteins and dramatically alter their structure and degree of compaction.