Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 183

2 Photoconversion of A. victoria GFP

2.1 Discovery and UV/VIS Spectroscopic Investigations
of Photoconversion of GFP

Photoconversion of GFPwas discovered shortly after the cloning of the gfp gene [ 1 , 7 ].
Photoconversion is found to occur in a range of different optical regimes, using UV
and visible light illumination, and with femtosecond and nanosecond pulses as well as
continuous illumination. The report by Chalfie et al. [ 1 ] on recombinant expression of
the gfp gene included a note: “Indeed, the fluorescence produced by 450- to 490-nm
light appeared to be more intense after brief photobleaching by 340- to 390-nm light”
[ 1 ]. A first spectroscopic characterisation of this photochromic reaction appeared in
1995 from the laboratory of Roger Tsien [ 7 ](Fig. 5 ) This paper showed the fluores-
cence changes with UV illumination at 280 nm. Cubitt et al. [ 7 ] proposed photo-
isomerisation as a mechanism and found the spectroscopic changes to be irreversible.
The fluorescence intensity changes in Fig. 5 indicate the occurrence of both photo-
bleaching and photoconversion under the conditions used, considering that at pH 8.0

a

4 q +5

0 min

3 q +5

13 min

2 q +5

23 min

40 min

1 q +5

0 q +0

325 350 375 400 425 450 475 500 525

Excitation wavelength (nm)

Fig. 5 First spectroscopic demonstration of photoconversion of GFP. Reproduced with permis-
sion from Cubitt et al. [ 7 ]. Selected figure caption: “Behavior of wild-type green fluorescent
protein (GFP) upon progressive irradiation. GFP samples were illuminated at 280 nm from a xenon
lamp and monochromator. Wild-type GFP suffered photoisomerization, decreasing the excitation
amplitude at 395 nm while increasing the amplitude at 475 nm. This effect was not reversible upon
placing the GFP in the dark” [ 7 ]