Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 149

none of these modes is perfectly localized, and in particular the C N and one of the

phenol ring modes show delocalization over the bridge and the phenol C-O bond,
respectively. In the anionic chromophore, the modes are the same, although the
frequencies are generally smaller, especially that of one of the two phenol modes.

¼

4.2 Dependence of Vibrational Modes on Chromophore Structure
and Its Interactions Inside Various Fluorescent Proteins

Not only the frequency but also the Raman and IR activities depend on the proto-
nation state of the chromophore. In addition, both the frequency and the activity are
modified by the protein matrix and depend on mutations in the chromophore or in
the chromophore environment. This makes the situation rather complex. However,
the studies of the last years suggest a key for the interpretation of the behavior
of these modes, which is summarized in Fig. 4 .
Considering for instance the C C mode (which can be easily identified as the

first high-frequency Raman active mode), its frequency is strictly related to the
C

¼

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C bond strength (and length). It is observed to decrease in frequency passing
from the neutral chromophore to the anionic one. This is related to the fact that the

Fig. 4 (a) Relationship between structure and mode frequencies for the first five high-frequency
fingerprint modes. The mode localization areas are described by the colored regions in the
structures on the left of the graph (for the mode description see also Table 1 ). The colored lines
in the graphs report qualitatively the mode frequency as a function of the resonance structure of the
molecule, modulated by the chromophore environment. (b and c) Mode frequency of the C

¼ C and

C N stretching modes ( green and magenta in panel a) as a function of the length of the

corresponding bond. Red , green , and blue dots correspond to RedFP, GFP, and BFP chromo-
phores, respectively. Filled dots correspond to neutral chromophores, empty dots to anionic
chromophores

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