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首页> 外文期刊>Journal of Molecular Biology >Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.
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Chaperonin-affected refolding of alpha-lactalbumin: effects of nucleotides and the co-chaperonin GroES.

机译：陪伴蛋白影响的α-乳白蛋白的折叠：核苷酸和陪伴蛋白GroES的影响。

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We have studied how nucleotides (ADP, AMP-PNP, and ATP) and the co-chaperonin GroES influence the GroEL-affected refolding of apo-alpha-lactalbumin. The refolding reactions induced by stopped-flow pH jumps were monitored by alpha-lactalbumin tryptophan fluorescence. The simple single-exponential character of the free-refolding kinetics of the protein allowed us to quantitatively analyze the kinetic traces of the GroEL-affected refolding with the aid of computer simulations, and to obtain the best-fit parameters for binding between GroEL and the refolding intermediate of alpha-lactalbumin by the non-linear least-squares method. When GroES was absent, the interaction between GroEL and alpha-lactalbumin could be well represented by a "cooperative-binding" model in which GroEL has two binding sites for alpha-lactalbumin with the affinity of the second site being tenfold weaker than that of the first, so that there is negative cooperativity between the two sites. The affinity between GroEL and alpha-lactalbumin was significantly reduced when ATP was present, while ADP and AMP-PNP did not alter the affinity. A comparison of this result with those reported previously for other target proteins suggests a remarkable adjustability of the GroEL 14-mer with respect to the nucleotide-induced reduction of affinity. When GroES was present, ATP as well as ADP and AMP-PNP were effective in reducing the affinity between GroEL and the refolding intermediate of alpha-lactalbumin. The affinity at a saturating concentration of ADP or AMP-PNP was about ten times lower than with GroEL alone. The ADP concentration at which the acceleration of the GroEL/ES-affected refolding of alphaLA was observed, was higher than the concentration at which the nucleotide-induced formation of the GroEL/ES complex took place. These results indicate that GroEL/ES complex formation itself is not enough to reduce the affinity for alpha-lactalbumin, and that further binding of the nucleotide to the GroEL/ES complex is required to reduce the affinity. Copyright 1999 Academic Press.
机译：我们已经研究了核苷酸（ADP，AMP-PNP和ATP）和伴侣蛋白GroES如何影响Gro-Apo-α-乳清蛋白对GroEL的影响。通过α-乳清蛋白色氨酸荧光监测由停流pH跳跃引起的重折叠反应。蛋白质自由重折叠动力学的简单单指数特征使我们能够借助计算机模拟定量分析受GroEL影响的重折叠的动力学痕迹，并获得最合适的参数用于GroEL与蛋白的结合非线性最小二乘法对α-乳白蛋白的中间体进行重折叠。当不存在GroES时，可以通过“合作结合”模型很好地表示GroEL与α-乳白蛋白之间的相互作用，其中GroEL具有两个α-乳白蛋白结合位点，而第二个位点的亲和力比α-乳清蛋白的亲和力弱十倍。首先，使两个站点之间存在负合作性。当存在ATP时，GroEL与α-乳清蛋白之间的亲和力显着降低，而ADP和AMP-PNP不会改变亲和力。该结果与先前报道的其他靶蛋白的结果比较表明，GroEL 14-mer在核苷酸诱导的亲和力降低方面具有显着的可调节性。当存在GroES时，ATP以及ADP和AMP-PNP可有效降低GroEL与α-乳清蛋白的复性中间体之间的亲和力。 ADP或AMP-PNP饱和浓度下的亲和力比单独使用GroEL时低约十倍。观察到GroEL / ES影响的αLA重折叠加速的ADP浓度高于核苷酸诱导的GroEL / ES复合物形成的浓度。这些结果表明，GroEL / ES复合物的形成本身不足以降低对α-乳清蛋白的亲和力，并且需要核苷酸与GroEL / ES复合物的进一步结合以降低亲和力。版权所有1999，学术出版社。

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