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bond length scheme changes passing from a benzenoid one, typical of the neutral
chromophore, to a mixed benzenoid-quinonoid, typical of the anionic structure
[ 38 ], where the exocyclic C
C bond has a mixed character (single-double). This
structural transition is also driven by the interaction of the chromophore with the
protein matrix. In fact, the hydrogen bonds, especially those on the phenolic
and imidazolidinone oxygens, influence the bond order of the chromophore. This
explains the range of frequencies that this mode assumes in different mutants of
GFP. In turn, this frequency mode could be used to give indications on the level of
bond length alternation present in the chromophore, and in particular to discrimi-
nate neutral from anionic forms of the chromophore. A similar behavior is found
also in the chromophores of different homologues of GFP (e.g., in orange and
red proteins [ 39 ]). This picture is confirmed by the observation that this mode
frequency is linearly related to the absorption frequency [ 36 ]: in fact, also this
property is related to the bond length alternation [ 38 ]. A very similar behavior
is found in the C
O mode [ 38 ], although this is more difficult to be used as
a fingerprint mode because it is not Raman active.
The three subsequent modes are more difficult to recognize and interpret because
their frequencies are very close and their character superimpose, especially in the
range of intermediate bond length alternation, in the anionic chromophore. How-
ever, they are also likely to decrease their frequency going toward the quinonoid
form, although with different velocities. In particular, the C
N mode is less
sensitive to the change in the bond length alternation because it preserves its double
bond character in both resonance forms. For this reason, since its frequency is
almost constant and its Raman activity quite high, it can be considered a reference
mode. Conversely, it is very sensitive to the presence or absence of the chromo-
phore tail stemming from the second post-translational oxidation in orange/red
proteins: its frequency is ~30 cm 1 smaller in the red proteins [ 41 ]. This could be
used to identify the occurrence of the second oxidation (Fig. 4 ).
Other modes in the range 1,000-1,500 cm 1 were considered, but in this region
the calculations are less accurate for the presence of many delocalized modes
at similar frequencies, which increases the difficulty in their description.
The lower frequency modes ( < 1,000 cm 1 ) were only more recently considered
due to the fact that they are less easily measured, and especially for the protein, they
need resonance Raman techniques. They correspond to vibrations delocalized
over the whole chromophore, less sensitive to the environment, but more sensitive
to the specific chromophore structure.
As an example, we analyze here two low-frequency modes that are clearly
visible in the Raman spectra of the chromophores and in the SERS spectra of the
proteins. All the other modes have small intensity, thus these two can be considered
the low frequency fingerprint modes of the chromophores. One is located at
~705 cm 1 in the neutral cis conformer of the chromophore [ 4 ] in water and organic
solvents. Its frequency is quite insensitive to the solvent and appears only slightly
shifted in different mutants of GFP (~710 cm 1 [ 13 ]) and in the blue and cyan
chromophores ([ 4 ] and Fig. 5b ). The comparison with theoretical DFT calculations
indicates that
is a largely delocalized planar vibration that
involves the
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