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excluded, therefore, Glu222 as the final receptor for the proton after
photoswitching; however, on a later paper, van Thor et al. found evidences of
decarboxylation of Glu222 upon photoconversion, on the basis of temperature-
dependent photoconversion and spectroscopy, X-ray crystallography, mass spec-
troscopy, and electron spin resonance spectroscopy experiments [ 48 ]. Different
from other photoswitchable fluorescent proteins, the authors could not observe any
cis-trans photoisomerization signature. Possible explanations are that this does not
happen in the specific mutant, or that the changes in the vibrational spectrum given
by a cis-trans isomerization are much smaller in intensity than the ones caused by
the deprotonation of the chromophore and therefore could not be observed.
Raman spectra were also studied to address the differences in the two metastable
states of a red RSFP, revealing a possible involvement of cis-trans isomerization
in the photoswitching process [ 47 ]. In order to assess the possibility that cis-trans
isomerization be a general feature of RSFPs, we investigated systematically vari-
ous chromophores, identified the fingerprints of their structural change, and then
searched these features in the Raman spectrum of the corresponding complete
folded proteins; in this way, a cis-trans isomerization has been demonstrated
as implicated in the photoconversion of at least two photoswitchable mutants of
the GFP (a blue one, BFPF, and a green-yellow one, EYQ1) [ 4 ].
As a first step, the photochromic behavior of three synthetic chromophores
in their neutral form, namely the analogues of (a) GFP, (b) Y66F GFP (BFPF),
(c) Y66W GFP (CFP) was studied [ 49 ]. Combined NMR, absorption and Raman
spectroscopy, completed by accurate theoretical calculations, demonstrated that
all the studied chromophores undergo the same cis-trans isomerization upon rever-
sible photoconversion [ 4 , 49 ].
In Fig. 5 , we show the Raman spectra of both cis and trans forms of the chromo-
phores of GFP (panel a, cGFP and tGFP, respectively), and, for the first time,
of CFP (cCFP for the cis form and tCFP for the trans form, panel c). Calculations
based on density functional theory for preresonant Raman spectra accurately
reproduce the experiments, except for a slight linear rescaling of the energy scale
[ 4 , 42 ] (Figs. 5c, d ); similar results were obtained for c/tBFPF [ 4 ]. These outcomes
allowed finding the spectral fingerprints of after cis-trans isomerization of the
chromophores. Based on this knowledge, we analyzed chromophore states in the
complete, folded protein in BFPF and EYQ1, by comparing the Raman spectra
of the photoconversion product with the ones of the chromophores. Our approach
Fig. 6 (continued) solid line), at pH ¼ 8, after the subtraction of the baseline; the asterisk
indicates an amide I mode. Ellipses in panels a and b with matching styles and colors emphasize
some fingerprint Raman peaks for the anionic chromophore that are present in the spectrum of both
the protein and the chromophore. (c-f) The dark red solid curves represent the Raman spectrum of
native EYQ1 at pH ¼ 4.7 after the subtraction of the baseline. For comparison are reported the
Raman spectra of the photoconverted form at pH ¼ 8( dark green solid curve ), and of neutral
cGFP and tGFP ( dark red and dark green dashed curves , same data of Fig. 5a ). Panels e and f are
magnifications of the highlighted regions in panels (c) and (d); the ellipses emphasize
corresponding fingerprint spectral regions for the cis-trans transition. Adapted with permission
from [ 4 ]. Copyright 2009 American Chemical Society
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