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Fig. 10 Conformation of the
EGFP chromophore upon
incorporation of (2-F)Tyr or
(3-F)Tyr. Interestingly,
crystal structure analyses
revealed only one
chromophore conformation in
case of EGFP[(2-F)Tyr]. In
contrast, in EGFP[(3-F)Tyr],
the fluorinated tyrosine
moiety can adopt two
conformations [ 17 , 57 ]. The
ratio of occupancy is 3:2.
These results correspond well
with the observations from
nonchromophore Tyr
positions ( vide supra )
Thus, the question a rose whether the two conformers result from flipping of the
Tyr moiety in position 66 like shown for Trp analogs ( vide infra )[ 58 ]. But until
now direct dynamic evidence could not be provided. However, it is more likely that
the two conformational states are a result of Tyr flipping during chromophore
formation and the residue is kind of frozen in the final chromophore in a statistical
manner [ 17 , 57 ]. In addition, even if by modeling and calculations some rotational
freedom was attributed to the chromophore, cis / trans photoisomerization can be
excluded as reason for the two states [ 63 , 64 ].
However, the appearance of only one chromophore conformer in EGFP[(2-F)
Tyr] must have sterical reasons since in this variant the chromophore has less
hydrophobic contacts with its vicinity and no interaction of the fluorine atom can
be assigned [ 17 ]. Indeed, crystal structure analysis revealed that after adoption of
the alternative conformation the fluorine atom would induce a direct clash with the
ring nitrogen of the chromophore.
For ECFP[(4-F)Trp], ECFP[(5-F)Trp] and ECFP[(6-F)Trp], 19 F NMR spectros-
copy confirmed that fluorinated Trp66 as a part of the ECFP chromophore exists
in two states (Fig. 11 ) and that the population of the states is temperature dependent
[ 58 ]. The slow exchange process was determined by 19 F NMR to occur in a time
frame of 1.2-1.4 ms and differences in enthalpy and entropy of the two states could be
measured. Chromophore isomerization is an unlikely process in EGFP ( vide supra )
and can be discarded as reason for the two states as well as different protonation states
or dimer formation. More likely, the different states originate from changes in the
vicinity of the chromophore, i.e., in its electronic micro-environment leading to
different shifts in the NMR spectrum (Fig. 11 ). As candidates, Tyr145 and His148
were identified. In GFP, His148 and Thr203 are in contact with the phenolic ring but
the
-barrel is distorted in the region of residues 144-150, which is not hydrogen
bonded to the adjacent backbone residues 165-170 [ 65 ]. By structure comparison, it
seems likely that Tyr145 and His148 can adopt two conformations in ECFP [ 11 , 58 ].
In the “major” conformation, Tyr145 points toward the protein interior, whereas
b
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