Gray T E, Fersht A R
MRC Unit for Protein Function and Design Cambridge IRC for Protein Engineering, Department of Chemistry, University of Cambridge, U.K.
J Mol Biol. 1993 Aug 20;232(4):1197-207. doi: 10.1006/jmbi.1993.1471.
The refolding of barnase in the presence of GroEL has been monitored on the millisecond to seconds time scale using stopped-flow kinetics. GroEL binds rapidly and tightly to the denatured enzyme with a second-order rate constant of greater than 1.3 x 10(8) s-1 M-1 and slows down greatly the rate of barnase refolding. However, addition of ever increasing concentrations of GroEL does not prevent barnase refolding completely, as would be expected from mass action if folding of barnase could proceed only in free solution. At saturating concentrations of GroEL, barnase refolds with a half-life of 30 s, compared with 50 ms for refolding of free enzyme. The rate-determining step in the refolding of free barnase is the reaction of a "late" folding intermediate. A mutant of barnase that fold more slowly (Ser-->Ala91), refolds at a correspondingly lower rate when bound to GroEL, suggesting that formation of the fully folded state may be rate limiting for folding on GroEL. For the slow-folding Ser-->Ala91 mutant, the rate-determining refolding step has a half-life of 180 ms. In sequential mixing experiments, a delay was introduced to allow the Ser-->Ala91 mutant to refold for 30 ms before being mixed with GroEL. This reduces by 50% the amount of mutant barnase initially bound by GroEL. As only 11% of this mutant barnase is fully refolded from the late intermediate in 30 ms, there is preferential binding of an earlier refolding state to GroEL. We show by single mixing experiments that binding, not hydrolysis, of ATP reduces the lag in regain of barnase activity seen with GroEL alone. In the presence of high concentrations of ATP and GroEL the rate constant for refolding of barnase approaches that found in their absence, probably because ATP reduces the affinity of GroEL for refolding barnase, such that bound barnase is released and refolds unhindered. The addition of exceedingly small quantities of GroES in the presence of excess GroEL and a moderate amount of ATP also has a marked effect on the barnase refolding rate constant, suggesting that GroES may have higher affinity for the barnase: GroEL complex than for GroEL.
利用停流动力学，在毫秒至秒的时间尺度上监测了在GroEL存在下barnase的重折叠过程。GroEL以大于1.3×10⁸ s⁻¹ M⁻¹的二级速率常数快速且紧密地结合到变性酶上，并极大地减缓了barnase的重折叠速率。然而，不断增加GroEL的浓度并不能完全阻止barnase的重折叠，而如果barnase的折叠只能在自由溶液中进行，根据质量作用定律这是可以预期的。在GroEL饱和浓度下，barnase重折叠的半衰期为30秒，而游离酶重折叠的半衰期为50毫秒。游离barnase重折叠的速率决定步骤是一个“晚期”折叠中间体的反应。一个折叠更慢的barnase突变体（Ser→Ala91），当与GroEL结合时，重折叠速率相应降低，这表明完全折叠状态的形成可能是在GroEL上折叠的速率限制因素。对于慢折叠的Ser→Ala91突变体，速率决定重折叠步骤的半衰期为180毫秒。在顺序混合实验中，引入了一个延迟，以使Ser→Ala91突变体重折叠30毫秒后再与GroEL混合。这使最初与GroEL结合的突变体barnase的量减少了50%。由于在30毫秒内只有11%的这种突变体barnase从晚期中间体完全重折叠，因此早期重折叠状态与GroEL有优先结合。我们通过单次混合实验表明，ATP的结合而非水解减少了单独使用GroEL时barnase活性恢复的延迟。在高浓度ATP和GroEL存在下，barnase重折叠的速率常数接近不存在它们时的速率常数，这可能是因为ATP降低了GroEL对重折叠barnase的亲和力，使得结合的barnase被释放并无障碍地重折叠。在过量GroEL和适量ATP存在下添加极少量的GroES也对barnase重折叠速率常数有显著影响，这表明GroES对barnase:GroEL复合物的亲和力可能比对GroEL的更高。
