The Fluorescence Dynamics of Single Molecules of Green Fluorescent Protein

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) × 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.