New aspects on the mechanism of GroEL-assisted protein folding

Petra Guhr , Sonja Neuhofen , Carol Coan , John G. Wise , Pia D. Vogel
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引用次数: 2

Abstract

The mechanism of assisted protein folding by the chaperonin GroEL alone or in complex with the co-chaperonin GroES and in the presence or absence of nucleotides has been subject to extensive investigations during the last years. In this paper we present data where we have inactivated GroEL by stepwise blocking the nucleotide binding sites using the non-hydrolyzable ATP analogue, (Cr(H2O)4)3+ATP. We correlated the amount of accessible nucleotide binding sites with the residual ATP hydrolysis activity of GroEL as well as the residual refolding activity for two different model substrates. Under the conditions used, folding of the substrate proteins and ATP hydrolysis were directly proportional to the residual, accessible nucleotide binding sites. In the presence of GroES, 50% of the nucleotide binding sites were protected from inactivation by CrATP and the resulting protein retains 50% of both ATPase and refolding activity. The results strongly suggest that under the conditions used in our experiments, the nucleotide binding sites are additive in character and that by blocking of a certain number of binding sites a proportional amount of ATP hydrolysis and refolding activities are inactivated. The experiments including GroES suggest that full catalytic activity of GroEL requires both rings of the chaperonin. Blocking of the nucleotide binding sites of one ring still allows function of the second ring.

groel辅助蛋白折叠机制的新进展
在过去的几年里,伴侣蛋白GroEL单独或与共同伴侣蛋白GroES复合以及存在或不存在核苷酸的情况下辅助蛋白质折叠的机制已经受到广泛的研究。在本文中,我们提供的数据表明,我们通过使用不可水解的ATP类似物(Cr(H2O)4)3+ATP逐步阻断核苷酸结合位点来灭活GroEL。我们将可接近的核苷酸结合位点的数量与GroEL的剩余ATP水解活性以及两种不同模型底物的剩余再折叠活性相关联。在所使用的条件下,底物蛋白的折叠和ATP水解与剩余的可接近的核苷酸结合位点成正比。在GroES存在的情况下,50%的核苷酸结合位点不被CrATP失活,得到的蛋白质保留了50%的atp酶和重折叠活性。结果强烈表明,在我们实验中使用的条件下,核苷酸结合位点具有加性,通过阻断一定数量的结合位点,可以使一定比例的ATP水解和重折叠活性失活。包括GroES在内的实验表明,GroEL的充分催化活性需要伴侣蛋白的两个环。阻断一个环的核苷酸结合位点仍然允许第二个环的功能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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