Kuwajima K, Makio T, Inobe T
Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan.
J Biol Phys. 2002 Jun;28(2):77-93. doi: 10.1023/A:1019993102869.
We studied the effect of GroEL on the kinetic refolding ofα-lactalbumin by stopped-flow fluorescence techniques. We usedwild-type GroEL and its ATPase-defficient mutant D398A, and studied thebinding constants between GroEL and the molten globule foldingintermediate at various concentrations of ADP and ATP. The results arecompared with titration of GroEL with the nucleotides, ADP, ATP-analogs(ATP-γS and AMP-PNP) and ATP, which have shown that bothADP and the ATP analogs are bound to GroEL in a non-cooperativemanner but that ATP shows a cooperative effect. Similarly, the bindingconstant between GroEL and the folding intermediate decreased in acooperative manner with an increase in ATP concentration although itshowed non-cooperative decrease with respect to ADP concentration. Itis shown that the allosteric control of GroEL by the nucleotides isresponsible for the above behavior of GroEL and that the observeddifference between the ATP- and ADP-induced transitions of GroEL isbrought about by a small difference in an allosteric parameter (the ratio ofthe nucleotide affinities of GroEL in the high-affinity and the low-affinitystates), i.e., 4.1 for ATP and 2.6 for ADP.
我们采用停流荧光技术研究了GroEL对α-乳白蛋白动力学重折叠的影响。我们使用了野生型GroEL及其ATP酶缺陷型突变体D398A，并研究了在不同浓度的ADP和ATP条件下GroEL与熔融球状折叠中间体之间的结合常数。将这些结果与用核苷酸、ADP、ATP类似物（ATP-γS和AMP-PNP）以及ATP对GroEL进行滴定的结果进行比较，后者表明ADP和ATP类似物均以非协同方式与GroEL结合，但ATP表现出协同效应。同样，GroEL与折叠中间体之间的结合常数随着ATP浓度的增加以协同方式降低，尽管相对于ADP浓度它表现出非协同降低。结果表明，核苷酸对GroEL的变构调控导致了GroEL的上述行为，并且观察到的GroEL在ATP和ADP诱导转变之间的差异是由一个变构参数（GroEL在高亲和力和低亲和力状态下核苷酸亲和力的比值）的微小差异引起的，即ATP为4.1，ADP为2.6。
