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state and leads to a remarkable charge separation in the excited state of the
GdFP chromophore. In contrast to ICT where a negative charge is (partially)
relocated, charge separation means that a neutral state is split up into a positive
and a negative charge which then are located at different positions in the chromo-
phore. This intramolecular charge separation causes changes in the electrostatic
potential around the chromophore. As a consequence, the protein matrix relaxes
toward new, more favorable electrostatic contacts between the chromophore
and its surrounding. Due to this structural relaxation, the energy level of the
excited chromophore is lowered, leading to a red-shift in fluorescence but not
absorbance, thereby causing the detected large Stokes shift (personal communi-
cation of Prof. M. Michel-Beyerle).
2.2.3 Appearance of GdFP Absorbance and Fluorescence Spectra
Within the av GFP family, the spectral features of ECFP are distinct because the
absorbance as well as the fluorescence spectrum consists of two clearly distinguish-
able maxima (i.e., spectral bands). In contrast, absorption and emission spectra of
GdFP consist of broad bands with no resolvable structures. In this context, it was
speculated that the double absorbance and fluorescence peaks of ECFP arise from
ground-state structural heterogeneity within the region between residues 145 and 149
or the chromophore itself (see Fig. 2c ). In the vicinity of the ECFP chromophore, two
structural conformations, named A 0 and B 0 , were indeed detectable in the crystal
structure. In contrast, GdFP did not show any heterogeneity in this respect; this was
postulated to be the main reason for its spectral homogeneity. However, recent
investigations on Cerulean GFP, an engineered mutant without ground-state hetero-
geneity, gave exactly the same double-humped spectra [ 20 ], and quantummechanical
calculations revealed that chromophore spectra in conformation A 0 and B 0 would be
identical [ 21 ]. In the context of these observations, Malo et al. gave a possible
explanation for the double-humped peaks. Taking into account that indole is in the
core of the chromophore structure, they argued that the complex indole photophysics
have been transmitted to the chromophore itself [ 20 ]. Namely, indole shows two
* electronic transitions 1L a and 1L b . Although this argumentation
sounds plausible, it does not explain the vanishing of the second absorbance and
fluorescence band in GdFP because (4-Am)Trp is a derivative of indole with the same
underlying electronic transitions [ 22 ].
p ! p
Influence of the Structural Context on Spectral Properties
The three-dimensional structures of ECFP and GdFP were found to be almost
identical (see Fig. 2c ). The most prominent novel interaction, which could be
identified from crystallographic distance considerations, was a slight shift of the
amino-chromophore of GdFP toward Phe165 (see Fig. 3 ). The two residues
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