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Another interesting mode is located at ~600 cm 1 and it is weak in the neutral cis
chromophore, absent in the neutral trans chromophore and very strong in the cis
anionic chromophore ([ 4 ], Fig. 5a , and black ellipses in Fig. 6a, b ). This mode is
observed in the mutant EGFP while it is very weak in the E 2 GFP [ 13 ], in agreement
with the fact that in these mutants the dominant forms of the chromophore are
anionic and neutral, respectively. The comparison with theory indicate that this
mode is a delocalized planar vibration with a deformation of both the rings and of
the C-O phenolic bond (it is shown in Fig. 1b ), which explains the strong variation
of the Raman intensity between neutral and anionic forms.
4.3 Raman Study of the Chromophore States in Photoswitchable
Reversibly switchable fluorescent proteins (RSFPs) were developed, which can
undergo repeated transitions between different states, e.g., bright and dark forms.
This property makes RSFPs particularly attractive as active labels in biological
systems for selective photolabeling applications or subdiffraction imaging.
The photophysical basis for this peculiar behavior, whose knowledge is needed
to guide the detailed tailoring of mutant properties, will be discussed in details
elsewhere in of this topic. Here, we will discuss mostly the vibrational spectroscopy
studies that shed light on the process. It is useful to remember that, in some fluo-
rescent proteins [ 43 - 45 ], X-ray crystallographic studies showed that the brightness
of these proteins is closely related to the conformation of the chromophore: the
chromophore in the dark state is in a trans configuration, while in the bright state it
shows a cis configuration. A similar mechanism was proposed as a general feature
for RSFPs in the family of GFP mutants [ 46 ]. X-ray studies of protein crystals,
however, are not applicable to proteins in solution or undergoing small confor-
mational changes upon irradiation, and cannot discern the protonation states of
chromophores and of other residues [ 47 ]. On the contrary, spectroscopic and in
particular Raman studies of chromophores while embedded in folded proteins
can provide detailed information on the chromophore structure, on its interactions
with the environment, and in general on the energy landscapes and the dynamics of
the processes involved [ 19 ].
The first vibrational study of the photoswitching in a GFP mutant (GFP uv , with
spectral and photoconversion characteristics very similar to wt GFP) has been
carried out by van Thor et al. using difference FTIR spectroscopy [ 2 ]. This work
demonstrated the impact of different protonation states of the chromophore in the
original and the photoconverted forms, and that there are only small changes in the
structural backbone of the protein, since only small changes in the amide I and II
spectral markers for protein conformational changes are detected; in this work, the
authors could detect no changes in any glutamic acid residue since they could not
observe any difference in the 1,710-1,760 cm 1 region (characteristic of a -COOH
mode) nor in the ~1,560 cm 1 region (characteristic of a -COO mode). The authors
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