Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa
Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa
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DOI：
10.1073/pnas.0500489102
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发表时间：
2005-07-05
影响因子：
11.1
通讯作者：
Hofkens, J
中科院分区：
文献类型：
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作者：
Habuchi, S;Ando, R;Hofkens, J
单个分子的可逆光开关已被证明为绿色荧光蛋白（GFP）的许多突变体。然而，迄今为止，在单分子水平上已经实现了有限数量的对光响应缓慢的开关事件。在这里，我们报告可逆的photoswitching特性中观察到的单个分子的Dronpa，一个突变的GFP样荧光蛋白，是从珊瑚Pectiniidae克隆。包封光谱表明，在488 nm处的强烈照射将Dronpa改变为暗淡的质子化形式，但即使在405 nm处的弱照射也将其恢复为明亮的去质子化形式。虽然Dronpa存在于酸碱平衡中，但只有光引发的质子化形式显示出开关行为。在单分子水平上，488和405 nm的光可以用来驱动分子在亮态和暗态之间来回移动。这种可逆的光开关可以重复> 100次。对辐射的响应速度几乎线性地依赖于辐射功率，响应时间为毫秒级。Dronpa光开关的完美可逆性使我们能够提出一个详细的模型，定量地描述了各种状态之间的相互转换。Dronpa对光的快速反应为活细胞中信号分子的快速扩散或运输带来了巨大的希望。
Reversible photoswitching of individual molecules has been demonstrated fora number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoincluced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated > 100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.