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首页> 外文期刊>Journal of Molecular Biology >GROEL-MEDIATED FOLDING OF STRUCTURALLY HOMOLOGOUS DIHYDROFOLATE REDUCTASES
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GROEL-MEDIATED FOLDING OF STRUCTURALLY HOMOLOGOUS DIHYDROFOLATE REDUCTASES

机译：结构均一的二氢叶酸还原酶的Groel-mediated折叠

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Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihydrofolate, reductases (murine DHFR, wild-type E. coli DHFR, and two E. coli DHFR mutants) in the presence and absence of the molecular chaperonin GroEL, We show that GroEL binds the unfolded conformation of each DHFR with second order rate constants greater than 3 x 10(7) M(-1)s(-1) at 22 degrees C. Once bound to GroEL, the proteins refold with rate constants similar to those for folding in the absence of GroEL, The overall rate of formation of native enzyme is decreased by the stability of the complex between GroEL and the last folding intermediate. For wild-type E. coli DHFR, complex formation is transient while for the others, a stable complex is formed. The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR. When complex formation is initiated from the native conformation, GroEL binds to a pre-existing non-native conformation, presumably a late folding Intermediate, rather than to the native state, thus; shifting the conformational equilibrium toward the non-native species by mass action. The model presented here for the interaction of these four proteins with GroEL quantitatively describes the difference between the formation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rate constant for the last folding step. Due to this kinetic partitioning, three different mechanisms can be proposed for the formation of stable complexes between GroEL and either murine DHFR or the two E. coli DHFR mutants. These data show that productive folding of GroEL-bound proteins can occur in the absence of nucleotides or the co-chaperonin GroEL and suggest that transient complex formation may be the functional role of GroEL under normal conditions. (C) 1997 Academic Press Limited. [References: 54]
机译：使用停止流荧光技术，我们检查了在存在和不存在两种结构同源二氢叶酸，还原酶（鼠DHFR，野生型大肠杆菌DHFR和两个大肠杆菌DHFR突变体）的重折叠和解折叠反应。分子伴侣蛋白GroEL，我们显示GroEL以22摄氏度大于3 x 10（7）M（-1）s（-1）的二级速率常数结合每个DHFR的未折叠构象。一旦结合到GroEL，蛋白质用类似于在不存在GroEL的情况下折叠的速率常数重新折叠。天然酶的总形成速率因GroEL和最后一个折叠中间体之间的复合物的稳定性而降低。对于野生型大肠杆菌DHFR，复合物的形成是短暂的，而对于其他大肠杆菌，则形成稳定的复合物。无论它们是由未折叠的还是折叠的DHFR形成的，稳定的络合物都是相同的。当从天然构象开始形成复合物时，GroEL结合到先前存在的非天然构象，大概是后期折叠中间体，而不是天然状态。通过质量作用使构象平衡向非天然物种转移。此处介绍的这四种蛋白质与GroEL相互作用的模型定量描述了瞬时复合物和稳定复合物形成之间的差异，该差异由释放和重新结合到GroEL的速率常数（相对于最后折叠步骤的速率常数）定义。由于这种动力学分配，可以提出三种不同的机制来在GroEL与鼠DHFR或两个大肠杆菌DHFR突变体之间形成稳定的复合物。这些数据表明，GroEL结合蛋白的生产性折叠可以在缺乏核苷酸或伴侣蛋白GroEL的情况下发生，这表明在正常条件下，短暂的复合物形成可能是GroEL的功能。 （C）1997 Academic Press Limited。 [参考：54]

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