Tsurupa G P, Ikura T, Makio T, Kuwajima K
School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113, Bunkyo-ku, Japan.
J Mol Biol. 1998 Apr 3;277(3):733-45. doi: 10.1006/jmbi.1998.1630.
We have analyzed the effect of the chaperonin GroEL on the refolding kinetics of staphylococcal nuclease and its three mutants by stopped-flow fluorescence measurements. It was found that a transient folding intermediate of staphylococcal nuclease was tightly bound to GroEL and refolded in the GroEL-bound state without releasing the non-native protein in solution, and the refolding rate in the GroEL-bound state was 0.01 s-1. The GroEL-affected refolding of the nuclease appears to be in decided contrast to that of apo-alpha-lactalbumin reported in our previous study, wherein alpha-lactalbumin was shown to be more weakly bound by GroEL and to refold in the free state in solution. In spite of the apparent difference between the proteins, the GroEL-affected refolding reactions of both the proteins can be represented by a common unified reaction scheme. On the basis of this scheme, the binding constant between the nuclease intermediate and GroEL was estimated to be larger than 10(9) M-1. The stoichiometry of binding of the nuclease and its mutants to GroEL was found to be two (nuclease/GroEL 14-mer). The increase in ionic strength resulted in a weakening of the interaction between the nuclease and GroEL, which was attributed to a weakening of the electrostatic attraction between the two proteins as a result of electrostatic screening by ions. Although ATP was found to accelerate the GroEL-affected refolding of the nuclease, the refolding rate was still far from the rate of the free refolding. The free refolding behavior of the nuclease and its mutants was restored in the presence of the cochaperonin GroES and ATP.
我们通过停流荧光测量分析了伴侣蛋白GroEL对葡萄球菌核酸酶及其三个突变体复性动力学的影响。结果发现，葡萄球菌核酸酶的一个瞬时折叠中间体与GroEL紧密结合，并在与GroEL结合的状态下复性，而不会在溶液中释放非天然蛋白，且在与GroEL结合状态下的复性速率为0.01 s-1。核酸酶受GroEL影响的复性似乎与我们之前研究中报道的脱辅基α-乳白蛋白的复性形成鲜明对比，在之前的研究中，α-乳白蛋白与GroEL的结合较弱，并在溶液中的游离状态下复性。尽管这两种蛋白质之间存在明显差异，但它们受GroEL影响的复性反应都可以用一个共同的统一反应方案来表示。基于该方案，核酸酶中间体与GroEL之间的结合常数估计大于10(9) M-1。核酸酶及其突变体与GroEL的结合化学计量比为两个（核酸酶/GroEL 14聚体）。离子强度的增加导致核酸酶与GroEL之间的相互作用减弱，这归因于离子的静电屏蔽作用使两种蛋白质之间的静电吸引力减弱。尽管发现ATP能加速核酸酶受GroEL影响的复性，但复性速率仍远低于自由复性的速率。在共伴侣蛋白GroES和ATP存在的情况下，核酸酶及其突变体的自由复性行为得以恢复。
