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by incorporation of NCAAs. In ds Red, the incorporation of (3-Am)Tyr increased
the QY from 0.04 to 0.16 and (3-F)Tyr introduction led to an increase from 0.04 to
0.25. The authors speculate that this increase may occur due to the higher bulkiness
of the fluorinated or aminated chromophores and that this higher bulkiness shields
the chromophore better from the surrounding of the molecule. Improper shielding
was dedicated to be the main reason for the low quantum yield of the monomeric
ds Red in comparison to its tetrameric form since in the monomer only one layer of
-strands is separating the chromophore from the solvent. However, it is difficult to
believe that a single fluorine- or amino-group could protect the chromophore from
the solvent influence in its cavity. To resolve these controversial issues, more
research has to be performed on these noncanonical ds Red variants (e.g., combina-
tion of guided evolution with NCAA incorporation).
2.5 Chromophores with Chalcogen Containing Trp Analogs
Sulfur and selenium as heteroatoms are known to induce physicochemical
changes, e.g., they are efficient static fluorescence quenchers. Unlike carbon,
nitrogen, and oxygen, selenium and sulfur atoms possess vacant d -orbitals in the
outer shell and can therefore act as electron acceptors. Thus, sulfur and selenium
containing systems should be more permissive to interact with nearby charges,
which might induce dipoles, additional dispersion forces, polarizabilities, exci-
plex formation, resonance energy transfer, or the formation of charge transfer
complexes [ 29 ].
To study the effects of chalcogen atoms in the chromophore, the Trp analogs
[2,3]Sep, [3,2]Sep, [2,3]Tpa, and [3,2]Tpa (see Fig. 1 ) were introduced into ECFP
[ 15 ]. No fluorescence could be detected, although the proteins were reported to be
properly folded [ 15 ]. Furthermore, absorbance around 400 nm was observed for all
ECFP variants indicating the presence of a chromophore. However, mass analyses
resulted in heterogeneous spectra without regular peak repetition, which was
explained by uncontrolled chemical reactions that occured during or after chromo-
phore formation.
In contrast, incorporation of [2,3]Sep, [3,2]Sep, [2,3]Tpa, and [3,2]Tpa (see Fig. 1 )
in EGFP (incorporation only takes place at position Trp57 outside of the chromo-
phore) resulted in clear mass spectra proofing incorporation. Interestingly, their
incorporation caused differences in fluorescence intensity when compared to
EGFP (see Table 1 ). This was not seen for any other Trp analog incorporation in
EGFP [ 11 , 15 ]; however, Trp57 is coplanar to the chromophore and interacts with it
by fluorescence resonance energy transfer [ 30 ]. 30% increase in fluorescence intensity
could be achieved by replacement of Trp57 by [3,2]Tpa [ 15 ]. It was argued that the
electronically rich sulfur/selenium species in a particularly favorable geometric
arrangement of the chromophore neighborhood facilitated the increase in absor-
bance and fluorescence intensities.
Indeed, upon introduction of Met and
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