Biology Reference
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5 FPs Spectra: Spectral Tuning by the Protein Environment
The sheer number of FPs discovered and engineered so far eludes an attempt of an
exhaustive review. Rather, this section will highlight the cases of two main families
of FPs, i.e., those containing a GFP-like or an RFP-like chromophore, focusing on
the structural mechanisms operating the spectral shifts. It is useful, before starting
to analyze the single cases, to bear in mind how the interaction between chromo-
phore and environment can influence light absorption and emission. The environ-
ment can act on the chromophore structure by deforming some bond lengths mainly
through hydrogen bonds (H-bonds), and/or by distorting the planarity of the chro-
mophore. In addition, it can differentially stabilize the ground and excited states by
electrostatic interactions. Recent experimental [
82
] and theoretical [
83
] studies
were devoted to understanding the tuning of spectral properties by the protein
matrix electrostatic field.
With regard to bond deformation, early Raman spectroscopy studies on the GFP
chromophore in solution and on some
av
GFP mutants revealed a tight correlation
between the shift in absorption peak and the frequency of a specific Raman-active
band [
17
]. This correlation was rationalized on the basis of the resonance structures
of the chromophore (depicted in Fig.
6
)[
84
,
85
], as arising from the selective
stabilization of one of the two resonance structures by the protein environment.
Variations observed in the Raman spectrum reflect a changing stabilization between
a benzenoid-like resonance structure (I in Fig.
6
) and a quinonoid-like resonance
structure (II). For example, the ubiquitous H-bond between the imidazolinone
carbonyl and conserved Arg at 96 (in
av
GFP numeration) increases the contribution
from the quininoid form (II), thus lowering the excitation energy. H-bonds to the
phenolate side, by contrast, tend to decrease the contribution of the quininoid form
in the anionic chromophore. Resonance structures are less accessible to the neutral
form of the chromophore, because the protonated phenol enforces a benzenoid-like
structure (the optical properties of the neutral chromophore are accordingly blue-
shifted with respect to the anion).
The same effects can be described in a complementary picture, recalling that
excitation involves charge displacement from the phenolate to the imidazolinone
and to the bridging carbon. H-bonds to the imidazolinone will preferentially
stabilize the excited state, and thus lower the excitation energy. Vice versa,
H-bonds to the phenolate will increase the excitation energy. Other electrostatic
interactions can be involved, particularly in the proximity of the bridging carbon,
another site of charge redistribution. The interplay among these factors can be such
that the final outcome becomes difficult to predict. Nonetheless, this general
scheme is useful to decipher the mechanisms of spectral tuning.
In the RFP chromophore, a third resonance structure is possible (Fig.
6
)[
79
],
thanks to the presence of the conjugated acylimine. H-bonds to this additional site
will tend to lower the excitation energy, stabilizing the excited state charge dis-
placement over the region.
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