Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 145

4 Results on Fluorescent Proteins and Their Chromophores

Since each frequency identifies a specific vibrational mode, which corresponds to
a specific pattern of displacement, vibrational spectroscopy can give structural
information. The modes localized on bonds have characteristic frequencies that
depend on the order of the bond (double, single) and on the ligands. For instance,
the C

O stretching mode has a characteristic frequency of ~1,700 cm 1 , the C C

bond of ~1,650 cm 1 . However, the fine tuning of the frequency depends on the
chemical environment of the bond. The situation for the delocalized modes is even
more complex, because their frequency depends on the specific structure of the
molecule. Comparison of the experiments with the theory, or with the results in
simplified models or isotope-substituted samples, allows on one side to link each
peak in a spectrum with a given vibrational mode, and on the other side to under-
stand the correlations between changes in the structure and in the vibrational
spectrum of a molecule.
In the following Sect. 4.1 , we report the state-of-the-art knowledge about the
vibrational properties of the GFP chromophores. In Sect. 4.2 , we will discuss how
the protein environment can affect the structure and the vibrational modes of the
chromophores in fully folded proteins. In Sect. 4.3 , the results on photochromic
fluorescent proteins epitomize the methods through which one can study the
relations between the function of the FP and structure of its chromophore using
vibrational spectroscopy; more results, based on time-dependent vibrational spec-
troscopies, will be reviewed in Sect. 4.4 .

¼ ¼

4.1 Identification of Vibrational Modes of Model Chromophores

The mode-identification process in the FP chromophores started over 10 years
ago and involved a number of theoretical, experimental, and mixed theoretical
experimental papers [ 4 , 9 , 13 , 35 - 41 ]. For the mode assignment, the comparison
between experiment and theory is fundamental. In fact, the experiment can identify
only the vibrational mode frequencies (i.e., resonances in IR or Raman spectra).
Some indications on the atoms involved in each mode can be extracted by the
frequency displacement in isotopically substituted molecules; however, the precise
pattern of displacement of a mode can be obtained only through the comparison
with theoretical studies. In particular, Raman experiments on synthetic analogues
of the GFP chromophore highlighted the differences in the vibrational spectra
linked to the different protonation states [ 36 ].
Tables 1 (a) and (b) report a summary of the assignment of the modes updated at
the current knowledge. There is an overall agreement on the assignment of the first
five modes in the range 1,700-1,530 cm 1 , also considered the fingerprint high-
frequency modes. In the neutral chromophore, they are assigned to bond-stretching
vibrations of C C, phenolic ring (two different

symmetries, in principle only one Raman active) and C

¼ O (only IR active), exocyclic C ¼

¼ N. The studies showed that