One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 17

Table 4 Excitation energies in eV (nm) of anionic RFP model chromophores
Method

p -HBMPI
ex. energy in eV
(nm)

p -HBMPDI
ex. energy in eV
(nm)

RFP chrom.
ex. energy in eV
(nm)

Expt. (gas) a 2.40 (521) a 2.26 (549) a -

2.26 (549) b 2.20 (564) c CASPT2 -

2.68 (463) d 2.39 (519) d 2.36 (525) e TD-DFT B3LYP

2.01 (616) f DFT MRCI - -

a Photodestruction experiments [ 59 , 63 ]
b CASPT2/CASSCF(12/11)/6-31 G(d), B3LYP geometry [ 72 ]
c

CASPT2/CASSCF(12/11)/6-31 G(d), CASSCF geometry, connections saturated with hydrogen
atoms [ 79 ]
d

TD-DFT B3LYP/631+G*, B3LYP geometry [ 71 ]

e

TD-DFT B3LYP/631+G*, B3LYP geometry, connections saturated with methyl groups [ 71 ]

f

DFT MRCI, B3LYP geometry [ 80 ]

blueshifted is the BFP chromophore (340 nm), whereas the most redshifted is the
uvKaede chromophore at 577 nm. Substantial variations do occur among different
proteins containing the same chromophore (see Sect. 5 ).
The systematic study allowed the authors to correlate the variation in spectral
properties with the extension of the

LUMO,
p ! p * character of the excitation is confirmed also for non-GFP chromophore
structures (HOMO, LUMO isodensities, and their difference are reported in Fig. 5 ).
Chromophores with a larger charge displacement, measured as the variation of
dipole moment between the HOMO and the LUMO, generally display a lower
excitation energy (as apparent from Fig. 5 , uvKaede involves a larger amount of
charge displacement than p -HBDI and RFP). This observation reframes quantita-
tively the idea that larger

p -conjugated system. The HOMO !

p -conjugated systems are associated with lower excitation

energies.
By minimizing the excited state structure at a planar conformation, it is possible
to calculate the emission energy. Such a kind of analysis was performed for p -HBI
and for the RFP chromophore using CASSCF/CASPT2, yielding values of 2.45 eV
(507 nm) [ 70 ] and of 1.76 eV (703 nm) [ 79 ], respectively. The first value is in good
agreement with the low temperature (77 K) fluorescence peak of a GFP chromo-
phore model 2 in ethanol (490 nm) [ 54 ]. Exploration of the excited state energy
surface reveals the presence of twisted intermediates and nearby conical-intersec-
tions seams [ 79 , 81 ] that are relevant to the photophysics of the chromophore, for
example explaining their very low emission quantum yield when not embedded in
the protein matrix.

2

The early study by Niwa et al. used ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxo-1-
imidazolacetate, a model chromophore with the methyl group in p-HBDI substituted with
CO2C2H5.