Studies of a thermophilic chaperonin system

嗜热伴侣蛋白系统的研究

基本信息

批准号：
7681384
负责人：
EDA KOCULI
金额：
$ 0.94万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-01 至 2008-10-15
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Protein folding is critical to the survival of all cells; it is the final step in the pathway from DNA to active protein. Proteins use various mechanisms to achieve their final folded state, including spontaneous folding and folding dependent on cellular machines known collectively as chaperones. The class of chaperones called chaperonins assists protein folding in an ATP-dependent manner and is responsible for folding many proteins, both newly synthesized and newly translocated across cellular membranes. They play a role in refolding heat or stress damaged proteins in the recovery from thermal or other insults. In this project, chaperonin-mediated protein folding by the GroEL-GroES system of the extreme thermophile, Thermus thermophilus, will be studied with the long-term goal of understanding how cellular systems have evolved to deal with environmental extremes and, more specifically, what the differences are between folding mechanisms in mesophilic organisms and those in thermophiles. The specific aims of this project are to: 1) identify potential substrate proteins for the thermophilic chaperonin, in addition to the 24 reported recently, by proteomic analysis of the protein occupants of the chaperonin complex recovered from T. thermophilus cells and evaluate both sets of proteins for their dependence on the chaperonin system for refolding in vitro, examining aggregation and chaperonin binding when diluted from denaturant, release by the addition of ATP and co-chaperonin, and recovery of native enzymatic activity or protein function; 2) investigate the ATPase and protein folding cycles of the T. thermophilus chaperonin system and determine the efficiency of refolding in terms of cycles of ATP hydrolysis required to fold a given protein, comparing this to the efficiency of the well-studied E. coli chaperonin system, both with homologous substrate proteins and with identical mesophilic or thermophilic proteins at the same temperature; and 3) initiate NMR studies of a thermophilic substrate protein bound to thermophilic GroEL, with the expectation that the ability to collect spectra at elevated temperature may provide new insights into the nature of the bound protein and its interaction with the chaperonin. Not only is the description of how proteins fold central to our understanding of how cells survive and grow, it is crucial to uncovering the causes behind diseases that result from protein misfolding. These include the amyloidoses, such as Alzheimer disease, prion diseases, such as "mad cow" disease, and certain genetic diseases, such as the most common form of cystic fibrosis. Understanding the processes of protein folding may lead to the uncovering of new drugs and new therapeutic approaches to these severe disorders. Page 2 Number pages consecutively at the bottom throughout Form Page 2

描述（由申请人提供）：蛋白质折叠对于所有细胞的生存至关重要；这是从 DNA 到活性蛋白质途径的最后一步。蛋白质使用各种机制来实现其最终折叠状态，包括自发折叠和依赖于统称为伴侣的细胞机器的折叠。称为伴侣蛋白的伴侣以 ATP 依赖性方式协助蛋白质折叠，并负责折叠许多蛋白质，包括新合成的和新跨细胞膜转运的蛋白质。它们在从热或其他损伤中恢复的过程中重折叠热或应激损伤的蛋白质中发挥作用。在该项目中，将研究极端嗜热菌（Thermus thermophilus）的 GroEL-GroES 系统的伴侣蛋白介导的蛋白质折叠，其长期目标是了解细胞系统如何进化以应对极端环境，更具体地说，了解什么是极端嗜热菌差异在于嗜温生物和嗜热生物的折叠机制之间。该项目的具体目标是： 1) 除了最近报道的 24 种之外，通过对从嗜热嗜热菌细胞中回收的伴侣蛋白复合物的蛋白质占据者进行蛋白质组学分析，鉴定嗜热伴侣蛋白的潜在底物蛋白，并评估两组蛋白质依赖伴侣蛋白系统进行体外重折叠，检查从变性剂稀释时的聚集和伴侣蛋白结合，通过添加 ATP 和辅助伴侣蛋白释放，以及天然酶活性或蛋白质功能的恢复； 2) 研究嗜热嗜热菌伴侣蛋白系统的 ATP 酶和蛋白质折叠周期，并根据折叠给定蛋白质所需的 ATP 水解周期确定重折叠效率，并将其与经过充分研究的大肠杆菌伴侣蛋白的效率进行比较系统，均具有同源底物蛋白和相同温度下相同的嗜温或嗜热蛋白； 3) 启动与嗜热 GroEL 结合的嗜热底物蛋白的 NMR 研究，期望在高温下收集光谱的能力可以为结合蛋白的性质及其与伴侣蛋白的相互作用提供新的见解。对蛋白质如何折叠的描述不仅对于我们理解细胞如何生存和生长至关重要，而且对于揭示蛋白质错误折叠导致的疾病背后的原因也至关重要。这些包括淀粉样变性，例如阿尔茨海默病、朊病毒疾病，例如“疯牛病”，以及某些遗传疾病，例如最常见的囊性纤维化。了解蛋白质折叠的过程可能会导致发现针对这些严重疾病的新药物和新治疗方法。第 2 页在整个表格第 2 页的底部连续编号页码