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of A. victoria GFP, Thr203 is replaced by tyrosine and, moreover, residue 65 is Gly
or Thr instead of Ser to promote ionization of the chromophore [ 66 ]. The Tyr203
hydroxyphenyl side chain is stacked on top of the phenol ring of the chromophore,
thereby effectively extending the delocalized
interactions. In fact, any aromatic residue at position 203 (His, Trp, Phe, and Tyr)
will increase the excitation and emission wavelengths by up to 20 nm, with the
extent of shift increasing in the stated order [ 5 ]. These mutants were rationally
designed, based on the crystal structure of S65T GFP, with the expectation that the
additional polarizability around the chromophore and
-electron system by
interactions would
reduce the excited state energy and, thereby, increase both the excitation and
emission wavelengths. The same design of a red-shifting chromophore environ-
ment has been found in nature in the yellow hydromedusan FP phiYFP [ 67 ].
The tetrameric eqFP611 displays the most red-shifted fluorescence emission of
any unmodified FP studied so far, with an emission peak at 611 nm (Fig. 4a ) that is
Stokes-shifted by 52 nm from its excitation peak at 559 nm [ 23 ]. The 2-imino-
methyl-5-(4-hydroxybenzylidene)imidazolinone chromophore formed from the tri-
peptide Met63-Tyr64-Gly65 assumes a coplanar trans configuration (Fig. 4b ),
stabilized by hydrogen-bonding interactions to Ser158 and Asn143 [ 68 ]. As yet,
eqFP611 is the only naturally occurring FP known to have a highly fluorescent trans
chromophore. For the dimeric variant d1eqFP611, we had previously noticed that a
red-shifted species can be enhanced by irradiation with pulsed 532-nm light, and
concomitant changes in the Raman spectrum suggested a trans-cis isomerization of
the chromophore [ 69 ]. Because the cis isomer is energetically preferred by an
isolated chromophore, we rationalized that destabilization of the trans conforma-
tion should result in a shift of the equilibrium conformation toward cis . Conse-
quently, we introduced the Asn143Ser substitution into d2eqFP611, so that one of
the two hydrogen bonds holding the phenolate moiety in the trans form is removed.
This highly fluorescent variant, denoted as d2RFP630, has its absorption (emission)
maximum shifted to the red by 24 (19) nm to 583 (630) nm (Fig. 4a ). The optical
spectra of d2RFP630 are significantly broader than those of eqFP611, suggesting
that the protein ensemble contains a mixture of spectrally distinct species. This
hypothesis was subsequently confirmed by X-ray crystallography (Fig. 4c )[ 70 ].
Encouraged by these findings, we anticipated a further destabilization of the trans
form and possibly a further red shift of the emission wavelength by weakening the
remaining hydrogen bond. Indeed, the mutation Ser158Cys caused an additional
red shift of the excitation (emission) to 588 (639) nm (Fig. 4a ), and the X-ray
structure confirmed that the chromophores were all in the cis conformation
(Fig. 4d ), stabilized by the hydrogen bond to Ser143 [ 70 ].
For chromophores for which the dipole moment increases significantly upon
optical excitation, the fluorescence emission often depends strongly on solvent
polarity and shifts to longer wavelengths during the excited-state lifetime (dynamic
Stokes shift). Boxer and coworkers have attributed the very large Stokes shift in the
far-red emitting DsRed variant mPlum to a picosecond solvation response [ 25 ].
This time-dependent shift in emission was not observed in its parental proteins,
implying that mPlum has a peculiar chromophore environment that allows such
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