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highlighted the relevance of Raman spectroscopy for the study of ground and
metastable states of optically active portions of proteins in solution, since Raman
is a nondestructive technique that allows monitoring on-the-flow the products of
photoconversion. Finally,
these results support
the hypothesis that cis - trans
isomerization is a general feature in RSFPs.
4.4 Time Resolved Vibrational Spectroscopy for Analyzing ESPT
Time-resolved vibrational spectra have been used to determine the proton pathways
in ESPT in various mutants of the GFP. The ESPT is explained in details in
elsewhere in this topic; here it is only necessary to recall that, upon photon-
absorption-driven excitation of the neutral A state of the chromophore into A * ,
this state is completely converted by deprotonation into the state I * in less than
200 ps; this state decays by fluorescence emission into the state I (which differs
from the anionic ground state B of GFP mutants for the position of some residues
close to the chromophore), which, mostly, rapidly reconverts to state A.
An example of differential time-resolved infrared (TRIR) spectra collected from
wild-type GFP by the group of Tongue [ 52 , 53 ] is shown in Fig. 7a . Negative peaks
correspond to modes of the protein that are present in the ground state, but not in
the excited state. Among these, some do not change within 200 ps, and are assigned
to modes localized on the neutral chromophore in its ground state (state A) by
comparison with the vibrational IR spectrum of a chemically synthesized GFP
chromophore analogue (HBDI) [ 52 ]. Some relatively small changes in the shown
spectrum were later [ 53 ] assigned to vibrational modes of A * , while no modes of I *
were observed, probably because their energies are shifted or their absorption cross
sections are low. However, in the shown data, there is a bleaching peak around
1,560 cm 1 (frequency typical for a mode of a glutamate -COO ), and another
peak appearing at 1,712 cm 1 (frequency typical for a mode of a glutamic acid
-COOH), within the same observed time scale. This behavior was not observed in
mutants that do not experience ESPT. Moreover, the time scale is exactly the same
for the fluorescence dynamics of the state A * . This fact demonstrates the connection
of the protonation of a glutamate residue with the transition from A * to I * . The
initial claim by Stoner-Ma et al . [ 52 ] that the involved glutamate is the E222 was
based on the known geometry of the chromophore environment and on comparison
with previous difference-IR spectra of the product of irreversible photoconversion,
where decarboxylation of E222 is involved. After this claim in 2005, several studies
have been carried out to provide finer details on the dynamics of GFP. In the same
year, van Thor et al. analyzed the similarities among the normal photocycle of GFP
and the irreversible photoconversion by combining X-ray crystallography, static IR
for intermediate photoreaction steps frozen at cryogenic temperatures, TRIR, and
smart mutagenesis [ 3 ]. Stoner-Ma and collaborators extended then their TRIR
measurements of wtGFP by recording polarization-resolved transient vibrational
spectra and applying the method to study proteins in which the chromophore
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