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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07408017/
• 关键词: Molecular chaperone; chaperone; GroEL; Folding mechanism; Globular proteins; alpha-Lactalbumin; Staphylococcal nuclease; シャペロン; スタフィロマッカルヌクレアーゼ; Molecular chaperone; chaperone; GroEL; Folding mechanism; Globular proteins; alpha-Lactalbumin; Staphylococcal nuclease; シャペロン; スタフィロマッカルヌクレアーゼ

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

为了理解体外蛋白质的蛋白质与生物细胞中的蛋白质折叠之间的关系，我们通过停止 - 氟蛋白（alphala）和葡萄球菌核酸酶（SNASE）的链蛋白凹槽对链球蛋白凹槽的影响。 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仅在过量的凹槽中观察到在凹槽结合状态下发生的缓慢重折叠过程。从离子强度的依赖性在凹槽存在下，重折叠反应的依赖性，上述差异下注……更多的是由于蛋白质静电性特性的差异所致。 α是一种酸性蛋白，在中性pH值为-7的净电荷为-7，而SNase是一种基本蛋白，净电荷为+12。另一方面，凹槽是一种强酸性蛋白，每月电荷为-20（-280每14mer）。因此，α和凹槽之间必须存在静电排斥以及SNase和Groel之间的吸引力。从本研究中，得出的结论是，远距离静电相互作用以及疏水相互作用以及疏水性相互作用对于groel识别靶蛋白非常重要可逆地绑定到凹槽，但也可以在凹槽结合的状态下重新折叠。结果表明，尽管凹槽对上述两个靶蛋白的重折叠反应的影响显然大不相同，但它们都可以用相同的统一反应方案来解释。较少的