Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 107

when compared to the parent protein. The other fluorinated variants, however,
exhibited a higher tendency for aggregation. This behavior was assigned to favor-
able or unfavorable effects of the fluorine atoms on the folding of the chromophore
surrounding protein matrix since EYFP[(2-F)Tyr] not only showed lower aggre-
gation tendency but also significantly higher extinction coefficients and relative
fluorescence. In contrast, EGFP[(2-F)Tyr] exhibits inferior spectral properties
(~25% decreased absorbance and fluorescence intensities) and a higher tendency
to aggregate.

2.4 Chromophores with (4-Am)Phe, (O-Me)Tyr and (3-Am)Tyr

2.4.1 GFPuv

Wang et al. reported site-directed substitution of Tyr66 of GFPuv with (4-Am)Phe
and (4-Me)Tyr [14] (see Fig. 1 ). They found that the absorbance (range: 375-435
nm) and fluorescence (range: 428-498 nm) maxima and quantum yields correlate
with the structural and electronic properties of the tyrosine substituents (see
Tables 1 and 2 ). Upon ( O -Me)Tyr incorporation, only one absorbance maximum
at 394 nm was observed, which is plausible since ( O -Me)Tyr66 does not provide a
protonation site. The fluorescence is blue-shifted and the quantum yield consider-
ably decreased (0.76

0.37). The fluorescence maximum can be detected at 460
nm, which fits well with the fluorescence maximum of the protonated form
of the av GFP chromophore (disappearing in a pico second timescale because of
ESPT [ 3 ]). Hence, this av GFP variant trapped the fluorescence of the protonated
chromophore form and might be a good model to study the spectral properties of
this chromophore species. Furthermore, (4-Am)Phe was introduced into the chro-
mophore and also exhibited a decreased yield in fluorescence. The GFP[(4-Am)
Phe], although blue-shifted (8 nm) when compared with the fluorescence emission

!

Table 2 Influence of phenyl ring substitution of residue 66 in the chromophore on excitation and
emission wavelength. The maximum wavelengths of both absorbance and emission peaks increase
with the electron-donating ability of the para substituents. The excitation maxima of Br and I
substituted chromophores (marked by asterisk ) do not fit exactly in this scheme. A possible
explanation is the interference of electron withdrawing effect and spin-orbital coupling (see text).
Substituent

Excitation wavelength/nm Emission wavelength/nm

-Br 375* 428

-I 381* 438

-H 360 442

-O-CH 3 394 460

-O-H 397 460

-NH 2 435 498

-O 475 506