The fluorescence dynamics of single molecules of green fluorescent protein
The fluorescence dynamics of single molecules of green fluorescent protein
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DOI：
10.1021/jp991968o
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发表时间：
1999-12-09
影响因子：
2.9
通讯作者：
Moerner, WE
中科院分区：
文献类型：
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作者：
Peterman, EJG;Brasselet, S;Moerner, WE
绿色荧光蛋白（GFP）的几种发黄光突变体的一个有趣的特性是它们在几秒的时间尺度上在荧光和非荧光状态之间切换。在聚丙烯酰胺凝胶中GFP突变体的单分子荧光研究中观察到这种特殊的闪烁行为（Dickson，R. M.;等人，Nature 1997，388，355）。主要利用黄色发光的酚盐阴离子突变体EGFP，我们报告新的单分子实验;研究几个参数对闪烁过程的影响：pH值，宿主基质和泵浦强度;在这些研究中的主要测量是观察到的开时间和关时间的分布。在6-10的范围内，EGFP的开启时间动力学与pH无关，因此使发色团的质子化/去质子化不太可能作为闪烁的来源。然而，激发强度对闪烁有相当大的影响：在高强度下，开启时间较短。我们将这些结果与系综漂白测量进行比较，系综漂白测量发现在pH 8的琼脂糖凝胶中EGFP的漂白量子产率为（8 +/- 2）x 10（-6）。每个光子吸收的单分子发射终止的概率与体漂白量子产率一致，从而表明这两个过程是相关的。
An interesting property of several yellow-emitting mutants of the green fluorescent protein (GFP) is that they switch between a fluorescent and a nonfluorescent State on a time scale of seconds. This peculiar blinking behavior was observed in single-molecule fluorescence studies of GFP mutants in poly(acrylamide) gels (Dickson, R. M.; et al. Nature 1997, 388, 355.). Utilizing primarily the yellow-emitting phenolate anion mutant EGFP, we report new single-molecule experiments; studying the effect of several parameters on the blinking process: pH, host matrix, and pumping intensity; The primary measurement in these studies is the observed distribution of on-times and off-times. The on-time dynamics of EGFP are independent of pH over the range of 6-10, thus making protonation/deprotonation of the chromophore unlikely as the source of the blinking. The excitation intensity, however, has a considerable effect on the blinking: the on-times are shorter at high intensity. We compare these results to ensemble bleaching measurements which find the bleaching quantum yield of EGFP in agarose gel at pH 8 to be (8 +/- 2) x 10(-6). The probability of termination of single-molecule emission per photon absorbed is in agreement with the bulk bleaching quantum yield, thus suggesting that the two processes are related.