Interaction of GroE with an All-B-protein*
Interaction of GroE with an All-B-protein*
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GroE 与全 B 蛋白的相互作用*
DOI：
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
Johannes BuchnerS
中科院分区：
文献类型：
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作者：
Marion Schmidt;Johannes BuchnerS
分子伴侣参与蛋白质在体内和体外的折叠。大肠杆菌伴侣GroEL与许多非天然蛋白质相互作用。GroEL识别的非天然蛋白质的共同结构基序尚未确定。为了研究β折叠二级结构在非天然蛋白质与GroEL相互作用中的作用，我们使用单克隆抗体的Fab片段作为模型底物蛋白。在这里，我们表明，GroEL相互作用的功能与此所有的蛋白质在重新激活。抗体片段从胍变性状态以良好的产率自发地重折叠。GroEL会短暂抑制功能性重折叠至天然状态，但在缺乏Mg-ATP和GroES的情况下，不会完全停止折叠。这些非特异性释放的GroEL结合的Feb片段的产率对应于在不存在伴侣的情况下自发再活化的产率。然而，在GroEL存在下的重折叠动力学是相当慢的。向GroEL-F复合物中加入Mg-ATP导致结合的底物蛋白立即释放，并且与自发再活化相比，重构抗体片段的量显著增加。GroES对于Fab片段的功能性GroEL介导的重折叠不是必需的，但在很小程度上影响重活化产率。有趣的是，GroEL介导的Feb重折叠的刺激主要取决于ATP、ATP-yS和AMP-PNP的水解。ATP酶活性的测量-为了研究无机磷酸盐的释放，如所述（34），在与铵离子和孔雀石绿绿色络合后用比色法测定。光散射-使用配备有磁力搅拌器的Perkin-Elmer荧光分光光度计MPF-44 A进行重折叠F. B片段的光散射。激发和发射波长设定为360 nm，狭缝为3 nm。在搅拌下记录聚集动力学。通过向再活化缓冲液中加入变性Fab片段开始反应。
Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative pro- teins, which is recognized by GroEL, has not yet been identified. In order to study the role of &sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the Fab fragment of a monoclonal an- tibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-&protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound Feb fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addi- tion of Mg-ATP to the GroEL-F, complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted an- tibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-me- diated refolding of the Fab fragment but affects the reactivation yield to a small extent. Interestingly, stim- ulation of the GroEL-mediated Feb refolding depends primarily on hydrolysis of ATP, ATP-yS, and AMP-PNP the Measurements of ATPase Actiuity-In order to investigate the release of anorganic phosphate was determined colorimetrically after complexation with ammonium molybdate and malachite green as described (34). Light Scattering-Light scattering of refolding F.b fragment was followed by using a Perkin-Elmer fluorescence spectrophotometer MPF-44A equipped with a magnetic stirrer. The excitation and emission wavelengths were set at 360 nm with slits at 3 nm. Aggre- gation kinetics were recorded under stirring. The reaction was started by adding denatured Fab fragments to the reactivation buffer.