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Gly67, Arg96 and Glu222. Nevertheless, for Tyr66 mutants and their noncanonical
variants, the fluorescence properties are preserved and even can be tremendously
changed as discussed above. We observed recently that Trp57 and Met218 in av GFP
play a crucial role for proper folding, chromophore maturation, and fluorescence [ 15 ].
For instance, Trp57 in av GFP is 15 ˚ apart from the chromophore and is not replaceable
with Leu [ 37 ]. However, its direct fluorescence is not detectable due to resonance
energy transfer to the chromophore [ 38 ]. In addition, Met218 cannot be replaced in
av GFP by another canonical amino acid without protein misfolding and loss of fluores-
cence [ 15 ].
3.2 Aliphatic NCAAs
First attempts to introduce 5 0 ,5 0 ,5 0 -trifluoroleucine (Tfl) or 6,6,6-trifluoromethio-
nine (Tfm) (see Fig. 1 ) into an EGFP mutant containing only two Met residues
(M78L, M88L, M153T, M233K) failed or gave only marginal incorporation effi-
ciency according to amino acid analysis [ 39 ]. In the EGFP-2M mutant, one Met
residue (M1) is solvent exposed, whereas the second (M218) is buried. Although
Met-containing species dominated over Tfm-containing variants, 19 F-NMR analy-
sis was performed. Thereby, two signals were detectable; both could be assigned to
the two different Tfm residues due to their different structural context. However,
the comparatively low expression yields of EGFP-2M[Tfm] might be caused by the
increased van der Waals radius of Tfm (84.4 ˚ ) compared to Met (71.2 ˚ )[ 40 ],
which disfavors its accommodation especially in the protein interior and/or causes
unfavorable interactions. In addition, such increase in size may interfere with a
proper recognition of the amino acid by the translational apparatus. This was
confirmed in recent studies by Tirrell and co-workers, who reported that trifluor-
onorleucine could only be incorporated into proteins after evolution of the methio-
nyl-tRNA synthetase, whereas norleucine is recognized at the same level as
methionine by the unmodified methionyl-tRNA synthetase [ 41 ].
Although EGFP-2M contains 19 Leu residues, ESI-MS analysis after incorpora-
tion experiments in the presence of Tfl revealed only species with a maximum of
5 substituted residues. In addition, a tremendously decreased expression yield was
observed [ 39 ]. Furthermore, amino acid analysis confirmed a Tfl content of only
< 7%. The increased size of the fluorinated amino acid might be the explanation for
this behavior, since the trifluoromethyl moiety has the size of an isopropyl group
[ 42 ]. Such increased bulkiness may be especially unfavorable in the context of the
fact that Leu is mainly distributed in the tightly packed protein core [ 42 ]. Therefore,
the accumulation of numerous local perturbations in the side chain packing proba-
bly induces significant disturbances in the protein core and subsequent unfolding of
the related GFP variant.
Nevertheless, Tirrell and co-workers succeeded in incorporating Tfl into a
mutant of GFPuv (Q80R, F99S, M153T and V163A) into which they included
two further mutations (S65G and S72A) [ 43 ]. However, the fluorinated GFP variant
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