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molecules similar to the chromophore, but with some changes in the structure itself,
in its environment, or in the isotopic composition of some of the atoms. Based on
this knowledge, it was possible to determine structural or protonation changes
responsible for the peculiar photophysics of some mutants of the GFP, as described
in Sects. 4.3 and 4.4 , or even in mutants of other families of autofluorescent
proteins; as a recent example, Raman spectroscopy allowed to link a reverse pH-
dependence of chromophore protonation with the large Stokes shift observed in the
red fluorescent protein mKeima [ 57 ].
Even more information can be achieved by single-molecule studies, which
are allowed also for vibrational spectroscopy by the SM-SE(R)RS techniques
( Sect. 2.2 ). Recently, Schleifenbaum et al. detected the same individual protein
and its photoproducts via fluorescence and SERS imaging [ 58 ]. By studying
single molecules, it was possible to assign distinct Raman bands to green and
red fluorescence forms of the studied bichromophoric autofluorescent protein
DsRed_N42H, revealing new insight into the photodegeneration processes of its
two chromophores [ 58 ].
As shown in Sect. 4.4 , also the dynamics of the chromophore can be studied by
time-resolved infrared and Raman spectroscopy; with still higher time resolution
(10 fs), achievable in future experiments, FSRS may allow even more detailed
views into structural changes in the dynamics along the chemical reaction coor-
dinate on the multidimensional potential energy surfaces of polyatomic molecules
[ 17 ]. Another time-resolved nonlinear technique, which could be applied in
the future for the study of the dynamics of FP chromophores, could be the
2-dimensional infrared (2D-IR) spectroscopy [ 59 ]. This can be considered an
extension of the 2D-NMR, but addressing the vibrational modes of a molecule.
The 2D-IR method allows to expose structural dynamics through the molecular
vibrations, similarly to the FSRS experiment discussed at the end of Sect. 4.4 , but
by considering the anharmonic coupling between different modes. The essential
advantages of 2D-IR are its ability to identify the different dynamic contributions
to the spectral shapes and their intrinsic time resolution. Furthermore, the 2D-IR
spectrum exposes directly not only the frequencies but also the anharmonicities of
vibrational modes, requiring a more quantitative interpretation of the spectrum.
It is also possible to study the time dependence of the coupling between
different vibrational modes. 2D-IR has alreadybeenappliedtostudypeptides,
proteins, and hydrogen-bond dynamics [ 60 ], and is therefore a good candidate for
vibrational-based structural and dynamical studies of proteins of the GFP family
as well.
Fig. 8 (continued) stretching and phenol-ring deformation modes. (c) Perspective view of the
phenol ring-wagging motion considered responsible for the oscillations mentioned above. Three
positions in the phenol-ring motion are illustrated to show how the phenol ring swings from the
native structure (I) toward (structure II) and possibly away (structure III) from the optimal ESPT
geometry; green background, protein pocket. All panels are adapted from [ 56 ], with permission;
copyright 2009 Nature Publishing Group
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