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h n A
h n
h n
h n
h n
Fig. 6 Outline kinetic model of the ESPT mechanism in avGFP
arising almost exclusively from the anionic form. Boxer and co-workers measured
the time-resolved fluorescence of avGFP with ultrafast time resolution [ 20 ]. The A*
state decays in a non-single exponential fashion with a mean lifetime of 18 ps. The
fluorescence spectrum of the anionic form was observed to grow in intensity on a
similar timescale of a few tens of picoseconds, with no change in spectral profile
[ 20 , 91 ]. This result clearly points to the occurrence of an ESPT reaction. Such
reactions are unique in biology, but have been well characterised in simpler
molecular systems [ 92 ]. The assignment to ESPT was confirmed by the observation
of a large deuterium isotope effect, which extended the A* state lifetime and
correspondingly increased the rise time for the green emission [ 20 ]. Similar obser-
vations were made using transient absorption spectroscopy [ 93 ].
Since the population of the anionic (B) ground state does not increase rapidly as
a result of irradiation, it is evident that the main fate of the deprotonated excited
state is decay (mainly radiative) followed by re-protonation to recover the A ground
state. Chattoraj and co-workers proposed a model which incorporates this beha-
viour (Fig. 6 ), where the emissive (deprotonated) state (called the I* state to
distinguish it from the directly excited ground state, B) is formed in the geometry
of the original ground state, and relaxes back to the A state. Ultrafast pump-dump-
probe spectroscopy revealed fast I
A proton-transfer dynamics on the ground
state surface which are sensitive to H/D isotope exchange [ 94 ]. It was proposed that
the B state is populated by a reorganisation of the protein matrix about I* occurring
with a low probability [ 20 ]. The X-ray structures of A and B states suggested that
the reorganisation involves T203 reorientation [ 95 ], and steady-state photochemi-
cal measurements show that an irreversible A
B conversion can be effected
photochemically, probably due to a low yield electron transfer and photodecarbox-
ylation mechanism [ 50 , 51 ].
The location of the proton acceptor was investigated by time-resolved vibrational
spectroscopy [ 96 - 99 ]. The transient infrared difference spectrum was monitored
following A state excitation with picosecond time resolution between 1500 and
1800 cm 1
(Fig. 7 ). The instantaneous appearance of four strong bleach bands
OD) is associated with excitation of the chromophore ground state
(cf. Fig. 3 ). This is accompanied by the immediate appearance of positive
signals due to vibrational modes in the excited state. These bands could be assigned
to specific vibrational modes by isotope labelling and polarisation studies of HBDI in
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