Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 158

has been isotopically labeled [ 53 ]. The results were compared both with the
polarization-resolved TRIR studies of the HBDI chromophore and its isotopes
and with quantum chemical calculations of the vibrational spectrum of HBDI.
The combination of these methods allowed them to assign most of the modes in
the complex transient IR spectrum of wtGFP. In particular, polarization-resolved
TRIR is able to measure the angle between the dipoles of the vibrational transitions
and of the electronic transition involved in the excitation of the system by the
actinic pump. Some of these data are shown in Fig. 7b, c , with the probe polariza-
tion oriented parallel ( D A ⊥ ) to the pump polarization. The

shown parameter D is obtained from the experimental data as:

A // ) and perpendicular ( D

¼ D

A ==
D

D

A ? :

From this, the angle between the two dipoles can be obtained as follows:

1 = 2

2 D

1

cos ðyÞ¼ :

D þ 2

Knowing this angle and the direction of the electronic transition dipole, there are
additional restraints to assign each peak to a given vibrational mode, whose dipole
direction can be estimated either because it is localized on a given bond or from
theoretical calculations. Already the observation of a complex behavior of the aniso-
tropy parameter within what could seem a single peak, gives the possibility to
discern among different contributions in the same spectral region. As an example,
the composite negative peak around 1,630 cm 1 has contributes from the bleach of
the ground state C C mode, the A * C

O mode, and possibly from a protein mode,
presumably a carbonyl or amide I vibration which is sufficiently close to the chromo-
phore to be influenced by the H-bond rearrangement which accompanies proton
transfer [ 53 ]. The final conclusion was the confirmation that the final step on the time
scale of 200 ps is the protonation of E222, without any evidence for any resolvable
kinetic, consistent with a concerted proton motion along a wire involving water
molecules and the S205 residue.
The same authors studied with similar techniques some blue-shifted mutants that
do not support ESPT [ 54 ], and some mutants of these where a much faster ESPT
with a different acceptor is recovered [ 55 ]. In all the studied cases, it was possible to
gain information on how electronic excitation can be coupled to specific structural
reorganization not only of the chromophore but also of the protein matrix. In the
end, this will help the rational design of new mutants with improved qualities, being
those a more stable A * state for a more efficient blue fluorescent protein, a faster

¼ ¼

Fig. 7 (continued) wtGFP, with parallel polarization in red and perpendicular polarization in
green . The anisotropy parameter ( D , see text) is shown in black with the right-hand axis: (b)4ps
after excitation; (c) 100 ps after excitation. All panels are reproduced with permission from [ 53 ];
Copyright 2006 American Chemical Society