Lin Z, Eisenstein E
Center for Advanced Research in Biotechnology, University of the Maryland Biotechnology Institute, Rockville, MD, 20850, USA.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1977-81. doi: 10.1073/pnas.93.5.1977.
The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction. The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin. The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates. The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1). The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding. The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex. The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides. These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin. The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.
大肠杆菌伴侣蛋白GroEL和GroES在ATP水解依赖性反应中促进多肽链的重折叠。在表面等离子体共振研究中，对多肽底物与GroEL结合和释放的基本步骤进行了研究，以测量枯草杆菌蛋白酶标准变体的结合和解离速率。确定的GroEL与此变体结合和解离的速率常数产生了一个微摩尔解离常数，这与独立的量热法估计结果一致。在5'-腺苷酰亚胺二磷酸（AMP-PNP）、ADP和ATP存在下，GroEL从非天然链上解离的速率显著增加，最大值在0.04至0.22 s(-1)之间。解离速率对AMP-PNP和ADP浓度的S形依赖性表明，多肽解离受核苷酸结合后发生的协同构象变化限制。释放速率对ATP的依赖性表现出两个S形转变，这归因于核苷酸与GroEL-多肽链复合物的远端和近端环面结合。添加GroES导致从GroEL释放非天然多肽的速率显著增加，表明共伴侣蛋白的结合比多肽的解离更快。这些数据证明了核苷酸结合促进的协同构象变化对于从GroEL释放链的重要性，这与伴侣蛋白对ATP的S形水解相关。根据伴侣蛋白作用单循环的工作假设，讨论了这些发现的意义。
