Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL.
Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL.
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核苷酸结合促进的构象变化从大肠杆菌伴侣蛋白 GroEL 中释放出非天然多肽。
DOI：
10.1073/pnas.93.5.1977
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发表时间：
1996
影响因子：
11.1
通讯作者：
Eisenstein,E
中科院分区：
文献类型：
--
作者：
Lin,Z;Eisenstein,E
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再折叠。在结合和释放多肽底物到GroEL的基本步骤进行了表面等离子体共振研究，以测量标准变异枯草杆菌的结合和解离率。确定了GroEL与该变体的关联和解离的速率常数，得到了微摩尔解离常数，与独立的量热估计一致。在5'-腺苷酸二磷酸（AMP-PNP）、ADP和ATP的存在下，GroEL与非天然链的解离速率显著增加，最大值在0.04 ~ 0.22 s（-1）之间。解离速率对AMP-PNP和ADP浓度的s型依赖性表明，核苷酸结合后发生的一致构象变化限制了多肽的解离。释放速率对ATP的依赖性表现为两个s型转变，归因于核苷酸与groel -多肽链复合物的远端和近端环面结合。GroES的加入导致GroEL中非天然多肽的释放率显著增加，这表明cochaperonin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一致构象变化对GroEL链释放的重要性，这与伴侣蛋白对ATP的s形水解有关。这些发现的含义是讨论在一个工作的假设，为一个单一的周期伴侣蛋白的作用。
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.