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It is interesting to note the presence of a Pro-rich PVPWP motif in GFPs with
unknown significance. It is speculated that the proline residues control the spatial
orientation of V55 and W57 as they play important roles in the protection of
the chromophore from collisional quenching by the solvent. Neither V55 nor
W57 can be substituted by another canonical amino acid without the loss of
fluorescence [ 37 , 45 ].
3.3 Aromatic NCAAs
3.3.1 Nonchromophore Positions
The chromophore of av GFP and its mutants requires an aromatic amino acid in
position 66 to exhibit fluorescence. Thus, the incorporation of noncanonical aro-
matic amino acids at this position opens the possibility to directly change the
structural and optical properties of the chromophore. The supplementation-based
incorporation (SPI) method [ 55 ] enables residue-specific replacement throughout
the whole protein structure. Therefore, structural consequences for all nonchromo-
phore position substitutions will be discussed first followed by a closer look to
structural effects of NCAAs upon their direct incorporation into the chromophore
GFP consists of 11 Tyr residues of which Tyr39, Tyr151, Tyr182, Tyr200 are
surface exposed, Tyr143 is partially exposed and Tyr74, Tyr106, Tyr145, Tyr92 are
buried in the protein core, whereas Tyr66 is part of the chromophore. Substitution
of all Tyr residues with either (2-F)Tyr or (3-F)Tyr led to small changes in
absorbance and fluorescence emission maxima as described above. Crystal struc-
tures of the two variants determined at 2.2 ˚ (EGFP[(2-F)Tyr]) and 1.6 ˚ (EGFP
[(3-F)Tyr]) [ 17 ] revealed that the overall fold was not affected by NCAA incor-
poration. Careful inspection of these and other known structures of fluorinated
proteins [ 56 ] revealed crystallographic distances between fluorine atoms and hydro-
gen donors allowing for weak interactions. However, fluorine cannot compete with
stronger hydrogen-bond acceptors such as oxygen and nitrogen. Interestingly, in all
positions, (2-F)Tyr exhibits only one conformation (see Fig. 7 ). None of the fluorine
atoms in the (2-F)Tyr residues of EGFP[(2-F)Tyr] is involved in particular inter-
actions [ 17 ]. In contrast, three (3-F)Tyr residues (92, 143, and 151) adopt two
conformations in EGFP[(3-F)Tyr] (Fig. 7 )[ 17 , 57 ]. The majority of these observa-
tions correspond well with the assumption that there is a correlation between
aromatic ring flipping and their burial in the protein's core. For example, (3-F)
Tyr145 is involved in various contacts with atoms of neighboring residues (Pro58
and His169) as well as water molecules and only one conformer is detectable.
Furthermore, the fluorine atom of (3-F)Tyr106 is in close contact (2.95 ˚ ) with the
ring carbons of Phe130. Surprisingly, buried (3-F)Tyr92 and partially exposed (3-F)
Tyr143 adopt both possible conformational states. The fluorine of (3-F)Tyr92 is in
interaction distance with the amide oxygen of Phe84 and most probably also with
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