Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 48

Fig. 4 Comparison of the
spectrum of neutral and
anionic HBDI in aqueous
solution with the spectrum of
avGFP

1.0

Protein
Neutral HBDI
Anionic HBDI

0.8

0.6

0.4

0.2

0.0

300 400 500

wavelength (nm)

Fig. 5 Electronic absorption
spectra of the HBDI anion in
a range of non-H-bonding
solvents

DiOx MeCN Ac DMF

1.0

0.8

0.6

0.4

0.2

0.0

360 380 400 420 440 460 480 500 520

Wavelength / nm

was reported that the spectrum matches the red-shifted protein absorption [ 53 , 54 ].
One interpretation of this is that the protein provides a vacuum-like environment
(although the medium must at least be polarisable). More recent measurements
have suggested that the red-shifted transition in the gas phase might arise from a
high multi-photon cross-section, and the one-photon absorption in fact peaks
further to the blue, as seen in solution [ 55 ].
Solvatochromism in HBDI has been studied extensively, partly in an effort to
reproduce the observed protein shift [ 56 , 57 ]. The largest spectral shifts are
observed for anionic HBDI, especially in polar non-H-bonding solvents such as
DMSO and DMF (Fig. 5 ). The latter result is interesting because this represents the
only solvent-charge combination for HBDI which comes close to reproducing the
large red-shift seen in the protein environment. The significance of this result is
unclear, as the solvent-induced shift is not accompanied by a significant increase in