3.3.2 NCAA in the Chromophore (Position 66)
The structure of the chromophore of av GFP and its mutants was subject of several
studies. For example, Ward et al. were the first documenting conformational
flexibility of the chromophore [ 63 ]. Chen and co-workers could demonstrate that
the chromophore cis / trans isomerization is not possible but the excited state could
adopt a twisted conformation. However, the chromophore is held in a strained
configuration by the surrounding residues, whereas its planarity (see Fig. 9 )isa
result of its delocalized
-electrons [ 64 ]. In this context, we discuss the effects of
aromatic NCAAs in position 66 to the structure of av GFP and its mutants.
As discussed above, incorporation of (2-F)Tyr and (3-F)Tyr results only in slight
changes of the spectral characteristics of EGFP and EYFP [ 17 , 57 ]. Noteworthy,
(2-F)Tyr and (3-F)Tyr have also different effects on the chromophore's structure
upon incorporation (see Fig. 10 ). Whereas the chromophore adopts only one
conformation in both variants of EYFP - mainly due to the Tyr203 which occupies
more space than the Thr203 of EGFP - the same holds true only for (2-F)Tyr in
EGFP since the chromophore including (3-F)Tyr shows two conformations [ 17 ,
57 ]. In general, structures of all crystals under study show a rather rigid architecture
for the chromophore and its surrounding as indicated by the B factors [ 57 ].
Furthermore, the phenolate oxygen of the chromophore interacts with Thr203 and
His148 and, in addition, the chromophore is in hydrophobic contact with Thr62,
Val150, and Phe165 [ 17 ].
Even if there are only slight differences in the occupancies of the two states of
EGFP[(3-F)Tyr] (distribution is approx. 3:2), one conformation is termed “major”
and the other one “minor”. Whereas in the “major” conformation the fluorine atom
is involved in a rather unusual interaction with C
2 of Thr203 (maybe weak -CF ...
HC- hydrogen bond), the “minor” conformation is characterized by a kind of
hydrogen bond between the fluorine atom and the hydroxyl group of Ser205 as
well as a conserved water molecule [ 17 , 57 ]. However, even with this information
the observed higher occupancy of the major conformer remains hard to explain.
Fig. 9 Electron density of the av GFP chromophore in planar ( left ) and top view ( right ). The
electron density reveals that the chromophore structure is nearly planar