MECHANISMS OF CHAPERONIN-ASSISTED PROTEIN FOLDING

伴侣蛋白辅助蛋白质折叠的机制

• 批准号: 2186891
• 负责人: Mark T Fisher
• 金额: $ 12.2万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2186891
• 关键词: adenosine diphosphate; adenosine triphosphate; alpha glucosidase; bioenergetics; chemical association; chemical binding; chemical kinetics; conformation; electron microscopy; glutamate ammonia ligase; hydrolysis; microcalorimetry; molecular chaperones; nucleotide analog; protein folding; protein sequence; proteolysis; solutions; stoichiometry; stop flow technique; thermodynamics; thiosulfate sulfurtransferase

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。