Examination of the folding of E-coli CspA through tryptophan substitutions
Examination of the folding of E-coli CspA through tryptophan substitutions
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DOI：
10.1110/ps.16201
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发表时间：
2001-10-01
期刊：
影响因子：
8
通讯作者：
Gregoret, LM
中科院分区：
文献类型：
--
作者：
Vu, DM;Reid, KL;Gregoret, LM
大肠杆菌冷休克蛋白，CspA，折叠非常迅速（时间常数，τ = 4毫秒）的一个明显的两个状态的机制。然而，最近的时间分辨红外（IR）温度跳跃实验表明，CspA的折叠轨迹可能更复杂。野生型CspA的唯一色氨酸（Trp11），用于通过荧光光谱法监测折叠过程，位于核酸结合位点内CspA表面上的不寻常芳香族簇中。为了更全面地了解CspA的折叠动力学，并确定是否存在任何先前未检测到的中间体，我们在蛋白质的三个不同表面位置引入了第二个色氨酸。这三个突变没有显著改变CspA的三级结构，尽管发现其中两个取代略微稳定。双态折叠，停流荧光光谱检测，是保留在所有三个突变体。这些结果表明CspA的快速折叠是由协同机制驱动的。
Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism. However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated. The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site. To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein. The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing. Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants. These results indicate that the fast folding of CspA is driven by a concerted mechanism.