maximum of GFPuv, had a Stokes shift of 63 nm, which makes it the second most
red-shifted noncanonical av GFP-based autofluorescent protein (GdFP has a Stokes
shift of 100 nm).
Comparing all generated noncanonical GFPuv variants from their study with the
spectral properties of neutral and anionic av GFP, Wang et al . found that the
maximum wavelengths of both absorbance and emission peaks increased with the
electron-donating ability of the para substituents [ 14 ]. However, Br and I substi-
tuted chromophores do not fit exactly in this row since they show red-shifted
excitation maxima in comparison to the nonsubstituted chromophore. This obser-
vation is difficult to explain since the electron withdrawing effect might interfere
with the spin-orbital coupling effects of halogen atoms as already discussed above.
With respect to applicability, Wang et al . argue that one of their generated
noncanonical mutants (GFP[(4-Me)Tyr]) could substitute BFP in the BFP-EGFP
FRET pair. This pair is not commonly used due to relatively low quantum yield
(0.24) and photostability of BFP. GFP[(4-Me)Tyr], however, has a higher quantum
yield (0.37) and good photostability, and would therefore significantly enhance the
applicability of the FRET pair with excitation possibility at ~400 nm and fluores-
cence detection at ~500 nm.
However, GFPuv[(4-Am)Phe] and GFPuv[(4-Me)Tyr], although already useful
for application, had considerably lower quantum yields than the original GFPuv.
To optimize the quantum yield of such noncanonical GFP variants, Sisido and co-
workers [ 28 ] reported a combination of in vitro incorporation of NCAAs and
random mutagenesis. Position S65 and Y145 were chosen for optimization since
these positions were already effective in improving the intensity of GFP and BFP
variants. They found that mutations Y145F, Y145L, and Y145M led to an increase
in fluorescence intensity up to fourfold. In this way, sequence optimization was
proposed to be a valuable method for optimization of unnatural autofluorescent
The generation of novel monomeric red fluorescent proteins is plausible as well.
For example, the introduction of a donor-acceptor type fluorophore such as ortho-,
or meta- amino-tyrosines should induce a large Stokes shift for the GFP chromo-
phore. The augmentation of a chromophore with an electron-donating group (amine
group) may result in even redder emission compared to GdFP (Prof. Hyundong
Yun, personal communication).
Recently, Goulding et al. incorporated (3-Am)Tyr (see Fig. 1 ) into ds Red-monomer
[ 16 ]. The incorporation caused a red-shift of 12 nm (603
615 nm) in fluorescence
emission, which is in high agreement to the proposed influence of electron donating
groups on the fluorescence wavelength maximum (see Table 1 ). Accordingly,
incorporation of (3-F)Tyr led to a blue shift in fluorescence maximum because of
the electron withdrawing effect of the Fluorine atom. In contrast to the experiments
performed with av GFP derived FPs, the quantum yield was significantly elevated