Photoactivatable GFP

Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297 1873-1877... 

Patterson, G. H. Lippincott-Schwartz, J. Selective photolabeling of proteins using photoactivatable GFP. Methods 2004, 32, 445 50. 

In the past two years, three new fluorescent protein variants have been generated that allow the selective activation or color conversion of fluorescent signal after specific illumination. The first variant, PA-GFP (photoactivatable GFP) [20] is based on wild-type Aequorea GFP, which has a bimodal absorption or excitation spectrum with two peak maxima, at 395 and 475 nm, corresponding to the protonated and the deprotonated states of the chromophore, respectively. When excited at 475 nm, wild-type Aequorea GFP emits maximal fluorescence at 503 nm, while excitation at 395 nm yields a maximum at 508 nm [2]. The latter large Stokes shift results from excited state deprotonation of the chromophore, as phenols become greatly more acidic in their excited states. Thus, excitation of the protonated chromophores gives emission at greater than 500 nm, similar to the direct excitation of the deprotonated chromophore. 

Recently, a photoactivatable variant from Aequoria victoria green fluorescent protein (pa-GFP) was reported (Patterson and Lippincott-Schwartz 2002), yielding an increase in fluorescence emission intensity (at k 520 nm) by a factor of 100 when excited at k 488 nm after spectral activation at A. 408 nm. This phenomenon is due to an internal photoconversion process in the protein and allows spectral photoactivation of this protein in a very local way such as in the nucleus of a living cell (Post et al. 2005). In tobacco BY-2 protoplasts, we transiently co-expressed pa-GFP or pa-GFP fusion proteins and red-fluorescent protein (DsRed)-tagged prenylated Rab acceptor 1 (Pral At2g38360), a membrane protein that localizes in speckles around the nuclear envelope. The DsRed transfection allows proper cell identification and visualization before activation (via Pral -DsRed fluorescence). After pa-GFP... 

FLAP (fluorescence loss after photoactivation) is a useful technique to investigate protein dynamics. The protein of interest can be fused to a photoactivatable green fluorescent protein (GFP) that upon irradiation increases fluorescence intensity around 100 times. Hence, after photoactivation of a certain cellular region, the protein diffusion rate can be determined by specifically tracking the photoactivated proteins [13]. 

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