Ranson N A, Burston S G, Clarke A R
Molecular Recognition Centre, School of Medical Sciences, University of Bristol, UK.
J Mol Biol. 1997 Mar 7;266(4):656-64. doi: 10.1006/jmbi.1996.0815.
Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 5% of the time taken for the substrate to commit to the folded state. The results imply that ATP drives the chaperonin complex through a cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.
线粒体苹果酸脱氢酶（mMDH）在存在GroEL、GroES和ATP的情况下比无辅助时折叠得更快。折叠速率随GroEL-ES浓度的增加在大约等摩尔的化学计量比（mMDH亚基：GroEL寡聚体）时达到最大值，并且GroE受体状态的表观解离常数K'至少为1×10⁻⁸ M。然而，即使在伴侣蛋白浓度为4000×K'时，即游离mMDH浓度可忽略不计的情况下，观察到的底物折叠速率仍保持在最佳状态，这不仅表明折叠发生在伴侣蛋白-mMDH复合物中，而且该速率不受与GroEL上位点的任何相互作用的抑制。尽管mMDH能够在伴侣蛋白上折叠，但捕获实验表明其在复合物上的停留时间仅为20秒。这与ATP周转速率以及GroES在复合物上的停留时间相关，并且仅约为底物进入折叠状态所需时间的5%。结果表明，ATP驱动伴侣蛋白复合物经历三个功能状态的循环：（1）一种受体复合物，其中未折叠的底物紧密结合；（2）一种封装状态，其中底物被隔离但失去了直接的蛋白质-蛋白质接触，从而使折叠能够不受阻碍地进行；（3）一种排出状态，它迫使底物解离，无论其是否已折叠。
