Molecular Mechanisms of Recognition of Target Proteins by the Chaperonin GroEL

伴侣蛋白 GroEL 识别靶蛋白的分子机制

基本信息

批准号：
07408017
负责人：
KUWAJIMA Kunihiro
金额：
$ 23.68万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

For the purpose of understanding the relationship between the protein refolding in vitro and the protein folding in a biological cell, we studied the effect of the chaperonin GroEL on the refolding kinetics of alpha-lactalbumin (alphaLA) and staphylococcal nuclease (SNase) by stopped-flow fluorescence spectroscopy. The results have shown that the effect of GroEL on the refolding reaction is apparently very different for SNase and apo-alphaLA.When the apparent refolding rate was estimated by measurements of the refolding reaction at different concentrations of GroEL,the refolding rate constant was changed in alphaLA,while the amplitude of the major kinetic process of the free refolding was decreased in SNase without large changes in the rate constants of the individual processes, and only the slow refolding process that occurred in the GroEL-bound state was observed in excess GroEL.From ionic-strength dependence of the refolding reaction in the presence of GroEL,the above difference bet … More ween the two target proteins was shown to be due to a difference in the electrostatic properties of the proteins. alphaLA is a acidic protein having a net charge of -7 at neutral pH while SNase is a basic protein with a net charge of +12. On the other hand, GroEL is a strongly acidic protein having a charge of -20 per monomer (-280 per 14mer). Therefore, there must be electrostatic repulsion between alphaLA and GroEL and attraction between SNase and GroEL.From the present study, it is concluded that the long-range electrostatic interactions as well as the hydrophobic interactions are important for the recognition of a target protein by GroEL.Next, we simulated the refolding processes of a protein under the influence of GroEL on a computer, on the basis of a scheme that the target protein is reversibly bound to GroEL but can also refold in the GroEL-bound state. The results have shown that although the effects of GroEL on the refolding reactions of the above two target proteins are apparently very different, they both can be interpreted in terms of the same unified reaction scheme. Less

出于蛋白质重折叠的蛋白质与生物细胞中蛋白质折叠的关系，伴侣蛋白凹槽在α-乳脂蛋白（alphala）的重折叠动力学上与停止流动荧光分类的重塑动力学对于snay和po-alphhala，重折叠反应上的凹槽一部分显然是非常不同的。估计在α的不同浓度的lopo g速率上估计重折叠反应的重折叠速率，而在alphala中则改变了重新折叠的主要动力学过程。在单个过程的E速率方面，在没有大的速率的情况下减少了snase，并且观察到在凹槽结合状态下发生的缓慢重折叠的PROCS，从而被观察到过多的凹槽。 ……死记因是蛋白质中蛋白质的静电性差异的差异。 20每个单体（每14毫升-280）因此，α和snase和groel之间必须存在吸引力。凹槽对计算机的影响的过程与凹槽可逆地结合，但在凹槽结合的状态下也可以重新折叠就相同的统一反应方案而言