Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer

描述(申请人提供)：对蛋白质在细胞环境中的折叠方式知之甚少。更具体地说，核糖体结合的新生蛋白质所达到的折叠程度和分子形状在很大程度上还没有被能够提供蛋白质构象直接评估的方法所探索。本研究的目的是研究在没有和存在触发因子(TF)伴侣的情况下，核糖体结合的新生蛋白的短程螺旋二级结构和疏水崩溃的程度。我们将研究新生链延长的不同阶段。来自三种蛋白质apoHmpH和Fim H的新生链将被分析。局部二级结构和最重要的是疏水塌陷是蛋白质体外折叠的两个主要驱动力。然而，当蛋白质从核糖体中涌出时，人们对它们在折叠过程中的重要性一无所知。这个项目将主要通过Forster共振能量转移(FRET)在受体不存在和存在的情况下通过测量FRET供体的荧光寿命来进行。我们将评估FRET效率的变化(与分子内距离分布的变化成正比)，以监测核糖体结合模型蛋白在新生链从核糖体隧道中出现时二级和三级结构的变化，以获得其局部结构和崩溃程度的证据。考虑到Tf非极性内表面可能有效地结合新生不完整蛋白质并显著改变其结构和致密化程度的事实，还将研究上述性质对Tf伴侣的存在和不存在的特定依赖性。

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.