MECHANISMS OF CHAPERONIN-ASSISTED PROTEIN FOLDING

伴侣蛋白辅助蛋白质折叠的机制

基本信息

批准号：
2701591
负责人：
Mark T Fisher
金额：
$ 10.19万
依托单位：
UNIVERSITY OF KANSAS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 2000-04-30
项目状态：
已结题

项目摘要

A number of in vivo protein folding and assembly reactions have been found to require an essential set of accessory proteins called chaperonins. Their mechanism of action is unknown. We will use dodecameric Escherichia coli glutamine synthetase (GS), mitochondria rhodanese, and yeast alpha-glucosidase as substrates for the E. coli chaperonin system, groEL and groES. Rhodanese requires all the chaperonin components and ATP for folding while GS and a-glucosidase only require groEL and ATP to initiate folding. Determining the molecular origins for these observed differences will provide important mechanistic information about chaperonin-assisted folding and assembly. The long range goal of our research is to define the mechanism by which the groE chaperonins increase the product yields of correctly folded oligomeric and monomeric proteins. The main hypotheses to be tested are that; 1) GroEL modulates its binding affinity for partially folded intermediates (PFI) by binding nucleotide (ATP, ADP, ATP analogs) and groES to rapidly release the previously bound substrate and; 2)the molecular origins of the different requirements of the chaperonin system observed with various substrates are dictated by a) nature and strength of binding of the initial folding intermediate and b) whether the release intermediate still has a tendency to misfold or aggregate. Our experimental approach is to; 1) determine the binding enthalpies and free energies between groEL and nucleotides, groES, and stable folding intermediates by differential stopped-flow titration microcalorimetry; 2) determine whether the chaperonin system is still required after the PFI has been released from immobilized (yet functional) groEL; and 3) determine the kinetics (monitoring time-dependent activity, fluorescence and enthalpic changes) of chaperonin-assisted folding (and assembly) reactions as a function of increasing concentrations of stable chaperonin-PFI complexes.

许多体内蛋白折叠和组装反应已经发现需要一组必需的辅助蛋白称为伴侣蛋白。他们的作用机理尚不清楚。我们将使用十二面体大肠杆菌谷氨酰胺合成酶（GS），线粒体大肠杆菌的罗丹尼人和酵母α-葡萄糖苷酶作为底物伴侣蛋白系统，凹槽和凹槽。罗丹人需要所有仅GS和A-葡萄糖苷酶折叠的伴侣蛋白成分和ATP 需要凹槽和ATP启动折叠。确定分子这些观察到的差异的起源将提供重要的机理有关伴侣蛋白辅助折叠和组装的信息。我们研究的远距离目标是定义该机制 Groe伴侣蛋白增加了正确折叠的产品的产量寡聚和单体蛋白。要测试的主要假设是那; 1）凹槽调节其对部分折叠的结合亲和力通过结合核苷酸（ATP，ADP，ATP类似物）和中间体（PFI）和 gros快速释放先前结合的底物； 2）伴侣蛋白系统不同要求的分子起源用各种底物观察到的是a）性质和力量初始折叠中间体的结合和b）是否释放中级仍然存在错误折叠或汇总的趋势。我们的实验方法是： 1）确定结合焓和凹槽和核苷酸，凹槽和稳定折叠之间的自由能通过差分停止流量滴定微钙化法的中间体； 2）确定是否仍需要 PFI已从固定（但功能性的）凹槽中释放出来； 3）确定动力学（监测时间依赖性活性，荧光和焓变的）伴侣蛋白辅助折叠（和组装）的变化反应是稳定浓度增加的函数伴侣蛋白-PFI复合物。