Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations
Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations
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DOI：
10.1073/pnas.0910246107
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发表时间：
2010-04-06
影响因子：
11.1
通讯作者：
Haran, Gilad
中科院分区：
文献类型：
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作者：
Frank, Gabriel A.;Goomanovsky, Mila;Haran, Gilad
分子伴侣GroEL至少以两种变构状态t和R存在，它们以ATP控制的方式相互转换。热力学分析表明，随着ATP浓度的增加，T态布居变得可以忽略不计，这与分子机器运行所必需的构象循环的要求相冲突。为了解决这个难题，我们对单环版本的GroEL进行了荧光相关光谱分析，使用了最近内置到其结构中的荧光开关，当添加ATP时，该开关会打开，即显著增强其荧光。测量了一系列相关函数作为ATP浓度的函数，并用奇异值分解进行了分析。分析将信号分配到两种动态明显不同的状态。令人惊讶的是，即使在ATP饱和的情况下，在任何时刻，大约50%的分子仍然填充在T状态，这表明T和R之间持续的不平衡循环。只有当添加辅伴侣素组时，T状态的粒子才会消失。我们的结果提出了一个模型，在该模型中，T/R比由ADP在水解后的释放速率控制，这可以相应地确定。
The molecular chaperone GroEL exists in at least two allosteric states, Tand R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, similar to 50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.