Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 47

a

b

0.3

0

-0.3

-0.6

-0.9

1660

1540 1580 1620 1700 1740

Wavenumber (cm -1 )

Fig. 3 (a) Simplified layout of pump-probe apparatus showing time delay between pump and
multi-wavelength probe. (b) Transient IR transmission spectrum recorded for HBDI in DMSO,
showing the bleach and recovery of the ground state accompanied by weak transient absorption.
Solid line 2 ps delay, short dash line 4 ps, long dash 6 ps, dot 10 ps. The ground state recovery is
fast, but included both excited state decay (see Sect. 3 ) and vibrational cooling in the ground state

a fraction in the anionic state (which can be formed photochemically [ 50 , 51 ]). The
neutral form absorbs to the blue of the anionic form, and in both cases the solution
spectra are significantly blue-shifted from the corresponding protein spectra. This
result points to a significant difference in electronic structure between the protein
bound and the free forms of the chromophore. It has been shown that the peak
absorption wavelength of GFP can be shifted significantly by mutations in the
surrounding amino acid residues, suggesting a strong sensitivity to the environ-
ment. For example, a number of mutants with the S65T mutation, the yellow
fluorescent proteins, show a large red-shift for the anionic form of the chromophore
[ 52 ]. Intriguingly, the spectrum of HBDI was measured in the gas phase, where it