Molecular Mechanisms of Recognition of Target Proteins by the Chaperonin GroEL

为了了解体外蛋白重折叠与生物细胞内蛋白折叠之间的关系，我们采用停流荧光技术研究了伴侣蛋白GroEL对α -乳清蛋白（alpha）和葡萄球菌核酸酶（SNase）重折叠动力学的影响。结果表明，GroEL对SNase和载脂蛋白α的再折叠反应的影响明显不同。通过测量不同浓度GroEL下的再折叠反应来估计表观再折叠速率时，alpha中的再折叠速率常数发生了变化，而SNase中自由再折叠的主要动力学过程的振幅减小，各个过程的速率常数没有大的变化，在过量GroEL中只观察到发生在GroEL结合状态下的缓慢再折叠过程。从GroEL存在下重折叠反应的离子强度依赖性来看，两种靶蛋白之间的上述差异是由于蛋白质的静电特性不同造成的。alpha是一种酸性蛋白质，在中性pH下净电荷为-7，而SNase是一种碱性蛋白质，净电荷为+12。另一方面，GroEL是一种强酸性蛋白质，每个单体的电荷为-20（每14mer的电荷为-280）。因此，alpha和GroEL之间一定存在静电斥力，SNase和GroEL之间一定存在静电吸引。从目前的研究中得出结论，远距离静电相互作用和疏水相互作用对GroEL识别靶蛋白很重要。接下来，我们在计算机上模拟了蛋白质在GroEL影响下的再折叠过程，基于目标蛋白质与GroEL可逆结合但也可以在GroEL结合状态下重新折叠的方案。结果表明，虽然GroEL对上述两种靶蛋白的重折叠反应的影响明显不同，但它们都可以用相同的统一反应方案来解释。少

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