Walter S, Lorimer G H, Schmid F X
Laboratorium für Biochemie, Universität Bayreuth, Germany.
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9425-30. doi: 10.1073/pnas.93.18.9425.
Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.
伴侣蛋白可防止蛋白质部分折叠或错误折叠形式的聚集，从而使其能够进行有效的折叠。有人提出，大肠杆菌的伴侣蛋白GroEL通过展开此类中间体来发挥这一功能，大概是以催化方式进行的。我们使用核糖核酸酶T1的还原和氨甲酰甲基化变体RCAM-T1作为底物，研究了GroEL诱导蛋白质展开的动力学机制。RCAM-T1无法完全折叠，因为缺少两个二硫键，因此它是长寿命折叠中间体的良好模型。添加GroEL时，RCAM-T1会展开，但GroEL不会改变展开的微观速率常数，排除了它催化展开的可能性。GroEL使RCAM-T1展开是因为它与蛋白质的未折叠形式具有高亲和力结合，从而将整体平衡向未折叠状态移动。当与GroEL结合的吉布斯自由能大于中间体的构象稳定性且其展开速率较高时，GroEL可通过这种热力学偶联机制展开部分折叠或错误折叠的中间体。
