Jackson G S, Staniforth R A, Halsall D J, Atkinson T, Holbrook J J, Clarke A R, Burston S G
Molecular Recognition Centre, University of Bristol School of Medical Sciences, U.K.
Biochemistry. 1993 Mar 16;32(10):2554-63. doi: 10.1021/bi00061a013.
Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.
为了追踪由核苷酸和cpn10结合引发的蛋白质结构重排，用芘马来酰亚胺标记了Cpn60。该共轭物分别以60%、60%和15%的芘荧光增强率结合ATP、AMP-PNP和ADP(P(i))。在每种情况下，结合都是协同的，半饱和（K1/2）分别出现在10微摩尔、290微摩尔和2500微摩尔，希尔常数（nH）分别为4、3和3。加入共蛋白cpn10会加强ATP、AMP-PNP和ADP(P(i))的结合，使K1/2值分别为6微摩尔、100微摩尔和<0.07微摩尔，并且协同性增加。用cpn10（7聚体）滴定cpn60/ADP（14聚体）复合物，亚基的化学计量比为14:7，证实了电子显微镜显示的分子不对称性。瞬态动力学表明，ATP最初与cpn60形成弱碰撞复合物（Kd = 4 mM），该复合物以180 s-1的速率异构化为强结合状态。我们认为，由ATP结合驱动的缓慢结构重排与伴侣蛋白对蛋白质底物亲和力降低是同一事件；在存在无结构多肽的情况下，AMP-PNP结合亲和力的丧失进一步支持了这一观点。因此，cpn60的这种重排类似于能量转导中的一个产生力的步骤。ATP水解的测量（pH 7.5，25℃）表明，与结构重排和产物解离相比，它是缓慢的（0.04 s-1）。这将稳态复合物定义为cpn60/ATP，这是伴侣蛋白的一种形式，它与底物蛋白的结合较弱。在结合未折叠的乳酸脱氢酶时，ATP水解速率提高了20倍，即使在没有cpn10的情况下，折叠酶的产量也会增加。加入这种共蛋白只会抑制cpn60中一半位点的水解，并导致折叠的LDH更快释放。提出了一种伴侣蛋白作用机制，该机制依赖于cpn60在对未折叠蛋白具有交替的低亲和力和高亲和力的状态之间循环。这个循环由ATP的结合和水解驱动。
