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Fig. 4 Influence of
chromophore fluorination on
fluorescence pH titration
profiles in parent and variant
EGFPs ( left ) and EYFPs
( right ). The pKa value of
EYFP[(3-F)Tyr] considerably
decreased by about 1.2 units,
while all other variants
showed only a change of
0.1-0.5 units
Fig. 5 pH dependency of absorbance and fluorescence of EYFP and its fluorinated variant EYFP
[(3-FTyr]. While EYFP absorbance is highly dependent on the pH (a), EYFP[(3-F)Tyr] absor-
bance has a strongly reduced sensitivity to pH (b)
Although the absorption spectra of native EGFP and EYFP were strongly pH
dependent, this tendency was less pronounced in the fluorinated variants (see Fig. 5 )
[ 17 ]. In EGFP[(2-F)Tyr], EGFP[(3-F)Tyr] and EYFP[(3-F)Tyr] significantly lower
tendencies for the occurrence of the non-fluorescing protonated chromophore forms
(absorbance at 395 nm) were detected. Among these variants, the decrease in pH to
a value of 4.5 had the smallest effect on EYFP[(3-F)Tyr], which is reasonable since
this variant also exhibited the lowest p K a when compared to the respective parent
protein EYFP. However, EYFP[(2-F)Tyr] did not follow the general tendency for
stabilization of the anionic chromophore state at low pH and behaved exactly like
the parent protein.
Additionally, the tendency of aggregation for all fluorinated EGFP and EYFP
variants was tested and an interesting correlation was found. Namely, there is
a direct relation between the appearance of the protonated chromophore species
at lower pH and the occurrence of aggregation after prolonged storage at 4 C.
Interestingly, the EYFP[(2-F)Tyr] exhibited a higher resistance to aggregation
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