Smith K E, Fisher M T
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160, USA.
J Biol Chem. 1995 Sep 15;270(37):21517-23. doi: 10.1074/jbc.270.37.21517.
Efficient renaturation of urea-denatured rhodanese using the chaperonin GroE system requires GroEL, GroES, and ATP. At high concentrations this renaturation also requires the substrate thiosulfate to have been present during GroEL-rhodanese complex formation. When thiosulfate is present the GroEL-rhodanese complex can be concentrated to greater than 1 mg/ml rhodanese with little effect on the efficiency of renaturation. However, if complex is formed in the absence of thiosulfate, renaturation of rhodanese in the presence of thiosulfate shows a critical concentration of approximately 0.4 mg/ml, above which renaturation yields drop dramatically. This critical concentration appears to be related to an aggregation event in the refolding of rhodanese. The nucleotide free or ADP-bound form of GroEL also binds to rhodanese that has been either already renatured or never denatured. The bound rhodanese has no activity but can be released from GroEL with ATP recovering 90% of control activity. The data presented herein support a release and rebinding mechanism for the GroE-assisted refolding of rhodanese. It also suggests GroEL binds several protein folding intermediates along the entire refolding pathway.
使用伴侣蛋白GroE系统使尿素变性的硫氧还蛋白高效复性需要GroEL、GroES和ATP。在高浓度下，这种复性还需要在GroEL-硫氧还蛋白复合物形成过程中存在底物硫代硫酸盐。当存在硫代硫酸盐时，GroEL-硫氧还蛋白复合物可以浓缩至硫氧还蛋白浓度大于1 mg/ml，而对复性效率影响很小。然而，如果在不存在硫代硫酸盐的情况下形成复合物，在硫代硫酸盐存在下硫氧还蛋白的复性显示出约0.4 mg/ml的临界浓度，高于该浓度复性产率会急剧下降。这个临界浓度似乎与硫氧还蛋白重折叠过程中的聚集事件有关。GroEL的无核苷酸或结合ADP的形式也会与已经复性或从未变性的硫氧还蛋白结合。结合的硫氧还蛋白没有活性，但可以通过ATP从GroEL中释放出来，恢复90%的对照活性。本文提供的数据支持GroE辅助硫氧还蛋白重折叠的释放和重新结合机制。这也表明GroEL在整个重折叠途径中结合几种蛋白质折叠中间体。
