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allowed us to identify the spectroscopic signatures of cis-trans isomerization and
separate them from those linked to different protonation states of the chromophore
[ 4 ].
With the help of Fig. 6 , we will review here, as a paradigm, the rationale of
the conclusion that, at pH
8, EYQ1 illuminated at 514 nm undergoes a photo-
conversion from a form with a cis anionic chromophore to one with a trans neutral
chromophore. More information about the materials and methods for this experi-
ment can be found in [ 4 ]; the absorption and fluorescence spectra of the various
forms of EYQ1 can be found in [ 50 ]. In Fig. 6b , the comparison between the Raman
spectra of the native and the photoconverted EYQ1 (blue and green solid lines,
respectively) demonstrates that the chromophore in the native form is deprotonated,
while the chromophore in the photoconverted form is protonated. This results from
the fingerprint-modes of the two chromophore forms discussed in the previous
section or shown in Tables 1a, b (some of those are highlighted by ellipses in
Fig. 6b ) and is confirmed by the comparison between the spectra of the protein
and of the chromophore with different protonation (Fig. 6a , blue dashed line for the
anionic chromophore, green dashed line for a neutral chromophore, in particular in
its trans form). Being in preresonance, indeed, the spectra in the protein are
characterized by the chromophore modes; as discussed in Sect. 4.2 , the small
differences observed in the spectra of the synthetic chromophores in solution with
respect to those inside the proteins stem from two factors: the protein backbone
(substituted in the chromophore by methyl groups where the backbone should
continue) and the different environment, i.e., a different network of hydrogen
bonds, electrostatic interactions and van-der-Waals interactions. As an example,
both contributions cause the weakening of some low-energy detected modes (solid
and dashed gray ellipses in Fig. 5a ), less (or not) visible in the noisier low-energy
part of the protein spectra; indeed, these changes were reproduced upon adding
elements of the protein environment in on-resonance TDDFT-based calculations,
after the identification of the corresponding modes in pre-resonance calculations.
Despite these differences, the assignment of the chromophore protonation is
clear from the data in Fig. 6a-b . The assignment of the stereoisomerization for the
photoconverted form, however, required a direct comparison with the Raman
spectrum of the protein in its native cis neutral A form, collected at pH
(Fig. 6d, f , solid dark-red curves). The last spectrum is indeed very similar to the
one of neutral cGFP (Fig. 6c, e , dashed dark-red curve), and the differences from
the spectrum of the photoconverted form are small and consistent with the ones
between neutral cGFP and tGFP. The enhanced red-shift after photoconversion of
the C
N mode (responsible for the highest intensity peak in Fig. 6f ) was present
also in the spectrum of photoconverted BFPF [ 4 ], and is most probably due to the
different environment in the protein pocket, since in the trans form the N atom is
more exposed to the protein environment, as shown for E 2 GFP by Nifos` et al . [ 51 ].
In conclusion of this section, Raman investigations allowed to demonstrate
that the cis-trans isomerization (Z-E diastereomerization) of the chromophore is
responsible for the photochromic properties of RSFPs in the GFP family, and to
discern the contribution of different protonation states. These investigations
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