Farr George W, Fenton Wayne A, Chaudhuri Tapan K, Clare Daniel K, Saibil Helen R, Horwich Arthur L
Howard Hughes Medical Institute and Department of Genetics, Yale School of Medicine, Boyer Center, 295 Congress Avenue, New Haven, CT 06510, USA.
EMBO J. 2003 Jul 1;22(13):3220-30. doi: 10.1093/emboj/cdg313.
Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL-GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL-GroES. Surprisingly, trans-only also folded smaller GroEL-GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a trans-only complex rescued a GroEL-deficient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL-GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more efficient folding.
尽管已经充分证明了GroEL介导的蛋白质折叠的顺式机制，即发生在由伴侣蛋白GroES封装的亲水性腔室内，但最近报道了GroEL - GroES介导的乌头酸酶（一种无法被封装的82 kDa大蛋白）的折叠过程。这个过程需要GroES与多肽相对的环（反式）结合，以驱动释放和有效折叠。在这里，我们使用仅反式复合物进一步评估了这一机制，其中GroES紧密连接到两个GroEL环之一，阻止该环结合多肽。在体外，仅反式复合物折叠乌头酸酶的动力学与GroEL - GroES相同。令人惊讶的是，仅反式复合物也能折叠较小的依赖GroEL - GroES的底物，如核酮糖-1,5-二磷酸羧化酶（Rubisco）和苹果酸脱氢酶，但速率比顺式反应慢。值得注意的是，在体内，编码仅反式复合物的质粒挽救了GroEL缺陷菌株，但菌落大小约为野生型GroEL - GroES产生的菌落大小的十分之一。我们得出结论，一种反式机制，即涉及与开放环的多轮结合并直接释放到本体溶液中，通常是有效的，尽管在尺寸允许的情况下，顺式封装支持更有效的折叠。
