The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation
The aggregation state of rhodanese during folding influences the ability of GroEL to assist reactivation
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DOI：
10.1074/jbc.m102500200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Horowitz, PM
中科院分区：
文献类型：
--
作者：
Bhattacharyya, AM;Horowitz, PM
罗丹明的体外折叠涉及正确折叠的酶的形成和非路径非活性物种之间的竞争。甘油或低温等共溶剂，例如10℃的复性，成功地延缓了大的非活性聚集体的旁路形成，但这一过程并不能产生100%的活性酶。这些数据表明，错误折叠的物种是由早期折叠中间体形成的。GroEL可以捕获早期折叠中间体，如果首先允许酶在提交给GroEL之前自发折叠更长时间，这一过程会导致非生产性中间体的形成，它就会失去捕获和重新激活若丹明的能力。此外，GroEL一旦形成大的聚集体就不能逆转，但它可以捕获一些折叠中间体并激活它们，即使它们如果留下来自发复性就不能形成活性酶。GroEL和罗丹尼斯之间的相互作用实质上但不是完全抑制了蛋白质内的失活，这是导致在无辅助复性过程中不完全激活的原因。因此，GroEL不仅减少了聚合，而且在所有辅助方法中提供了最高的重新激活。结果用以前提出的基于罗丹尼斯(Gorovits，B.M.，McGee，W.A.和Horowitz，P.M.(1998)Biochim BiPhys)的自发折叠研究的模型来解释。学报1382,120-128和Panda，M.，Gorovits，B.M.和Horowitz，P.M.(2000)J.生物化学275，63-70)。
The in vitro folding of rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 degreesC, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim Biophys. Acta 1382, 120-128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. BioL Chem 275, 63-70).