2.3 Chromophore Halogenations
2.3.1 Trp and Phe Halogenations
David Tirrell and co-workers incorporated position 6 halogenated Trp analogs in a
CFP variant (CFP6; F64L, S65T, Y66W, V163A) [ 13 ], the laboratory of Peter
Schultz incorporated para substituted Br- and I-phenylalanine [ 14 ] (see Fig. 1 )in
GFPuv and our group studied the effects of fluorinated Trp analogs on the chromo-
phore spectral properties of ECFP [ 15 ].
Generally, halogenation of Trp and Phe in the av GFP chromophore leads to a blue
shift in fluorescence. Tirrell and associates reported a 14 nm blue shifted fluorescence
for CFP6[(6-Cl)Trp] and CFP6[(6-Br)Trp] when compared to CFP6 (see Table 1 ).
Schultz and co-workers found exactly the same blue shift in fluorescence for GFPuv
[(4-Br)Phe] and GFPuv[(4-I)Phe] when these two variants are compared to a class 7
GFP with phenylalanine in the chromophore. A direct comparison to GFPuv is not
reasonable since a high red-shift in fluorescence wavelength occurs because of
inhibition of ESPT for (Br)Phe and (I)Phe substituted chromophores. Monofluorina-
tion of Trp at position 4-7 also caused slight blue shifts in fluorescence between 2 and
10 nm in ECFP [ 15 ]. The same tendency was also found when the Tyr residue in
ds Red was exchanged to (3-F)Tyr (See Sect. 2.4.2.) [ 16 ].
Quantumyields (QYs) of the halogenated av GFP variants were determined as well.
While incorporation of (6-Cl)Trp and (6-Br)Trp into CFP6 yielded ~60% reduction
in quantum yield when compared to CFP6, (4-Br)Phe and (4-I)Phe caused ~98%
reduction when introduced into GFPuv. This effect was explained by the influence of
halogens on the
-electron system according to the spin-orbital coupling theory [ 24 ].
In other words, halogens promote intersystem crossing, which leads to radiationless
decay of the excitation energy. QYs were not measured for the fluorinated ECFP
variants. However, the same effect can also be seen in the relative fluorescence when
compared to ECFP, where all variants exhibit significantly lower fluorescence inten-
sities. Noteworthy, the strength of the effect was dependent on the position of the
indole ring substitution, which was found in halogenated porphyrins as well [ 25 ].
Finally, the low quantum yields of the brominated and iodinated GFPuv and
CFP6 might also be attributed to sterical effects caused by halogen presence since
Br and I have very high van der Waals volumes [ 26 ], which might not be well
accommodated in the av GFP structure. In line with this argumentation, CFP6 does
not have the mutations N146I and M153T, which lead to better spectral properties
by enhancing the accommodation of the Trp-containing chromophore ( vide supra ).
2.3.2 Tyr Fluorination
EGFP and EYFP
In the context of GFP spectroscopy, the use of Tyr analogs is not only a suitable tool
to study the influence of certain functional groups on the GFP's spectral properties,