Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 105

e.g., intensity of absorbance and fluorescence, and blue/red-shifts in the emission
maxima. It also bears the potential to modify the anion/neutral equilibrium of the
Tyr's hydroxyl group because electron donating or withdrawing groups have an
influence on the acidity of the -OH group. In EYFP, it has to be considered that a
global replacement of Tyr by its analogs not only leads to a modified chromophore
but also to a modified Tyr203 in the vicinity (stacking position), which also will
have influence on the spectral properties of the chromophore.

Spectral Properties

Global substitution of all Tyr residues by (2-F)Tyr (see Fig. 1 ) in both, EGFP and
EYFP, resulted in protein variants with slight blue shifts (6-10 nm) in the absor-
bance as well as in the fluorescence maxima of their chromophores (see Table 1 ).
This is consistent with the findings of Trp fluorinations in ECFP (see above). In case
of EGFP[(3-F)Tyr], however, the absorbance spectrum is 3 nm blue shifted,
whereas the fluorescence maximum is slightly red-shifted (4 nm). Conversely, in
EYFP[(3-F)Tyr] both, a red-shifted absorbance and fluorescence was found.
Although it is difficult to unambiguously identify a possible origin of this spectral
behavior, one has to take into account the regioisomeric differences between the
fluorinated Tyr residues. For example, different positioning of fluorine atoms in the
phenol ring of Tyr certainly influences electron delocalization, the orientation of
transition- and ground-state dipoles. Studying the role of protonation on protein
spectral properties, we found uniform behavior of all fluorinated variants of EGFP
and EYFP at low (pH 1) or high (pH 11) pH. At low pH, when only the protonated
form of the chromophore is present and the protein is acid denatured, all variants
exhibited the same absorbance as EGFP or EYFP (l max ¼ 378 nm) [ 17 ]. At high pH

(only deprotonated form of the chromophore is present), the protein variants had
equally blue-shifted absorbance maxima when compared to ECFP or EYFP (blue
shift of 3 nm to a l max of 444 nm). Obviously, the main reason for the spectral
differences between these variants in the folded state are sterical effects caused by
the fluorination at different positions of the phenol side chain of the Tyr residues.

Influence of Tyr Fluorination on Chromophore pKa

The influence of the ring substituents on the p K a of the Tyr side chain could indeed
be detected in variants of EGFP and EYFP. The decrease in p K a in the fluorinated
variants was reported to be in the following order: parent protein

(2-F)Tyr-

>

protein (3-F)Tyr-protein (see Fig. 4 ). This corresponded well with the p K a values

of the free amino acids, Tyr (10.0)

>

(3-F)Tyr (8.5) [ 27 ].
Interestingly, the p K a value of (3-F)Tyr-EYFP considerably decreased by about
1.2 units, while all other variants showed only a change of 0.1-0.5 units. This was at
least partly attributed to the stacking interactions between (3-F)Tyr66 and (3-F)
Tyr203.

(2-F)Tyr (9.04)

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