Analysis of De Novo Protein Folding by Fluorescence Resonance Energy Transfer

通过荧光共振能量转移分析从头蛋白质折叠

基本信息

批准号：
8852633
负责人：
Silvia Cavagnero
金额：
$ 27.98万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852633
关键词：
Affect Alzheimer&apos s Disease Amber Amino Acids Amyotrophic Lateral Sclerosis Anticodon Automobile Driving Back Binding Biomedical Research Cells Charge Codon Nucleotides Complex Data Dependence Disease Electron Microscopy Energy Transfer Engineering Environment Evolution Fluorescence Fluorescence Resonance Energy Transfer Foundations Goals Health Human Huntington Disease In Vitro Knowledge Lead Length Light Measurement Messenger RNA Methodology Modeling Molecular Molecular Chaperones Molecular Conformation Monitor National Institute of General Medical Sciences Parkinson Disease Pattern Positioning Attribute Prevention Property Protein Conformation Proteins Research Ribosomes Sampling Shapes Solutions Staging Structure Surface Testing Transfer RNA Translations United States National Institutes of Health Variant Work apomyoglobin base crosslink disease diagnosis driving force experience fluorophore molecular shape polypeptide premature protein folding research study

项目摘要

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.

描述（由申请人提供）：关于蛋白质在细胞环境中折叠的方式知之甚少。更具体地说，核糖体结合的新生蛋白实现的折叠程度和分子形状在很大程度上没有通过能够直接评估蛋白质构型的方法来探索。这项研究的目的是在不存在和存在触发因子（TF）伴侣的情况下研究核糖体结合的新生蛋白的短距离螺旋二级结构和疏水性塌陷的程度（或缺乏）。将检查新生链伸长的不同阶段。将分析源自三种蛋白质Apomyoglobin，ApoHMPH和FIM H的新生链。局部二级结构，最重要的是疏水性崩溃是蛋白质折叠的两个主要驱动力。但是，随着蛋白质从核糖体中出现时，在折叠的背景下的重要性一无所知。该项目将主要由Forster共振能量转移（FRET）通过荧光寿命测量在不存在和存在受体的情况下进行。我们将评估FRET效率变化（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的二级和三级结构的变化，因为新生链从核糖体隧道中出现，以获得有关其局部结构和崩溃程度的证据。鉴于TF非极性内表面可能有效地结合了新生的不完整蛋白质并极大地改变其结构和程度，因此也将研究上述特性对TF伴侣存在的特定依赖性。