One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 14

initially neglected. Recently, a model for the neutral chromophore was devised by
adding a methyleneammonioum cation to p -HBDI, charging the molecule at a site
sufficiently detached from the conjugated system. The spectrum of this model
compound peaks at 399 nm [ 65 ], close to absorption of the neutral band of av GFP.
How well this model describes the neutral chromophore was discussed in [ 66 ].

4.1.2 Solution

With respect to gas-phase experiments, absorption of p -HBDI in water is blue-
shifted and exhibits a rather broad band peaking at 425 nm [ 17 , 57 ] (see Table 2 ).
In nonpolar solvents, the absorption maximum is at 440 5 nm. Increased solvent

polarity yields redshifted values. The longest wavelength peak, 482 nm, was
recorded in pyridine. The wavelength shift in going from water to pyridine is rather
sizable (57 nm) and corresponds to an energy shift of 2,755 cm 1 (0.34 eV). Using
the multivariant Kamel-Taft fit on the data in these various solvents, one can
extrapolate a value of 437 nm (2.84 eV) for the gas-phase, i.e., a solute with zero
acidic, basic, and polar parameters [ 57 ].
The significant solvatochromic shift of the anionic state points to a marked
sensitivity to the surrounding environment. Such sensitivity emerges also when
the chromophore is embedded in the protein matrix: in different FPs, the absorption
corresponding to the anionic chromophore shows a similar, if not larger, range of
variation (see Sect. 5.1 ). Absorption of neutral and cationic p -HBDI peaks at 368
and 393 nm respectively, and displays a much narrower solvatochromism (variation
of ~20 nm, around 1,000 cm 1 ).
Besides the main band, reported in Table 2 , the absorption spectra of both neutral
and anionic p -HBDI reveal features at shorter wavelength (around 300 and 250 nm
for p -HBDI and at 320 nm for p -HBDI) [ 55 , 57 ]. Similar blueshifted bands are also
measured in AHBMI (model chromophore of asFP and KFP) [ 50 ] and in model
chromophores of BFPF, BFP, [ 55 ], and Kaede [ 60 ]. Features around 330 nm are
also detectable in red FPs [ 67 ]. In av GFP, by contrast, these excitations merge with
the absorption of other aromatic residues in the protein. Excitation to these states is
characterized in some cases by strong two-photon cross-section (see Sect. 6 ).
As stated above, solvated model chromophores are generally poorly fluores-
cent at room temperature [ 54 , 68 ]. For instance, the chromophore fluorescence
quantum yield in DMF (dimethylformamide) is 0.00005 [ 60 ]. More appreciable
fluorescence is instead displayed by AHBMI and by uvKaede chromophore
models in DMF solution, with fluorescence quantum yields of 0.0021 and
0.005, respectively [ 50 , 60 ].

4.2 Chromophores of FPs: Computational Studies

Quite a number of theoretical/computational studies were dedicated to the optical
spectra of model chromophores of FPs in the gas phase [ 66 , 69 - 76 ]. Attempts to