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microscopy. The only disadvantage of this system is the relatively broad emission
peak, whereas the quantum yield is similar to ECFP [ 70 ].
It should be mentioned again that most commonly used FPs usually have poor
brightness when illuminated with UV light
366 nm. In order to address this
general limitation, Skerra and co-workers [ 73 ] recently combined in a FRET system
the fluorescence activity of a FP with the spectrally matching fluorescence of a
NCAA translated into the protein during its expression. In particular, they suc-
ceeded in incorporating site-specifically (~20 ˚ away from the protein's fluoro-
phore) the fluorescent (7-hydroxycoumarin-4-yl-)ethylglycine in ECFP. They chose
ECFP as the FRET acceptor for this NCAA because its absorption maximum in the
range of 430-450 nm overlaps with the 7-hydroxycoumarin emission maximum
( l max approx. 450 nm). Expectedly, the ECFP modified in this way exhibited
efficient FRET between its two fluorophores, having 'cyan' emission at 476 nm
upon excitation in the near-UV at 365 nm.
Finally, new classes of autofluorescent proteins could be generated using
NCAA. For instance, we could recently demonstrate that a single Trp
Trp substitution turned colorless anxA5 into a blue fluorescent protein [ 74 ]. The
same effects are known for (6-Aza)Trp and (7-Aza)Trp but these chromophores
suffer either from lower biocompatibility or fluorescence intensity compared to
(4-Aza)Trp [ 75 ]. In the future, we will explore the utilities of designing autofluor-
escent proteins by using fluorescent amino acids as building blocks for protein
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