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首页> 外文期刊>Journal of Molecular Biology >Kinetic Analysis of ATP-dependent Inter-ring Communication in GroEL.
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Kinetic Analysis of ATP-dependent Inter-ring Communication in GroEL.

机译：GroEL中依赖ATP的环间通信的动力学分析。

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摘要

Different concentrations of ATP were mixed rapidly with single-ring or double-ring forms of GroEL containing the Phe44-->Trp mutation and the time-resolved changes in fluorescence emission, upon excitation at 295 nm, were followed. Two kinetic phases that were previously found for double-ring GroEL are also observed in the case of the single-ring version: (i) a fast phase with a relatively large amplitude that is associated with the T-->R allosteric transition; (ii) and a slow phase with a smaller amplitude that is associated with ATP hydrolysis. In the case of weak intra-ring positive cooperativity, the rate constant corresponding to the T-->R allosteric switch of single-ring GroEL displays a bi-sigmoidal dependence on ATP concentration that may reflect parallel pathways of the allosteric transition. The slow phase is absent when double-ring or single-ring forms of GroEL are mixed with ADP or ATP without K(+), and it has a rate constant that is independent of ATP concentration. A third fast phase thatis still unassigned is observed for both single-ring and double-ring GroEL when a large amount of data is collected. Finally, a fourth phase is observed in the case of double-ring GroEL that is found to be absent in the case of single-ring GroEL. This phase is here assigned to inter-ring communication by (i) determining its dependence on ATP concentration and (ii) based on its absence from single-ring GroEL and the Arg13-->Gly, Ala126-->Val GroEL mutant, which is defective in inter-ring negative cooperativity. The value of the rate constant corresponding to this phase is found to increase with increasing intra-ring positive cooperativity, with respect to ATP. This is the first report of the rate of ATP-mediated inter-ring communication in GroEL, in the presence of ATP alone, which is crucial for the cycling of this molecular machine between different functional states.
机译：将不同浓度的ATP与单环或双环形式的含有Phe44-> Trp突变的GroEL迅速混合，并跟踪在295 nm激发时荧光发射的时间分辨变化。在单环版本的情况下，还观察到了双环GroEL的两个动力学相：（i）与T-> R变构转变相关的振幅较大的快速相; （ii）和振幅较小的慢相，与ATP水解有关。在弱环内正协作性的情况下，与单环GroEL的T-> R变构开关相对应的速率常数显示出对ATP浓度的双S形依赖性，这可能反映了变构过渡的平行路径。当双环或单环形式的GroEL与不带K（+）的ADP或ATP混合时，不会出现慢相，并且它的速率常数与ATP浓度无关。当收集大量数据时，对于单环和双环GroEL都观察到仍然没有分配的第三快速阶段。最后，在双环GroEL的情况下观察到第四阶段，而在单环GroEL的情况下发现没有第四阶段。通过（i）确定其对ATP浓度的依赖性和（ii）根据其不存在单环GroEL和Arg13-> Gly，Ala126-> Val GroEL突变体的情况，将此阶段分配给环间通讯。环间负协作性有缺陷。发现相对于ATP，对应于该阶段的速率常数的值随着环内正合作性的增加而增加。这是在单独存在ATP的情况下，GroEL中ATP介导的环间通讯速率的首次报道，这对于该分子机器在不同功能状态之间的循环至关重要。

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