FAST EVENTS IN PROTEIN-FOLDING INITIATED BY NANOSECOND LASER PHOTOLYSIS
FAST EVENTS IN PROTEIN-FOLDING INITIATED BY NANOSECOND LASER PHOTOLYSIS
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DOI：
10.1073/pnas.90.24.11860
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发表时间：
1993-12-15
影响因子：
11.1
通讯作者：
EATON, WA
中科院分区：
文献类型：
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作者：
JONES, CM;HENRY, ER;EATON, WA
通过光引发蛋白质折叠可以显着提高动力学研究的时间分辨率。在这里，我们展示了一个通过使用还原细胞色素 c 的血红素-一氧化碳复合物的纳秒光解来光触发折叠反应的示例。光学触发基于这样的观察：在不稳定条件下，细胞色素c可以通过一氧化碳优先结合到未折叠状态下的共价连接的血红素基团来展开。因此，一氧化碳的光解引发了折叠反应。我们使用时间分辨吸收光谱来监测血红素的结合。在折叠开始之前，我们观察到来自未折叠多肽的非天然和天然配体在微秒时间尺度上的瞬时结合。动力学模型表明，蛋氨酸-65 和-80 的分子内结合比组氨酸-26 和-33 更快，尽管组氨酸更接近血红素。这种光学触发应该为研究细胞色素 c 中的链折叠和二级结构形成提供一种强大的方法，而不受时间分辨率的任何限制。
Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are closer to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.