Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 53

Proton
Transfer

A*

Rare

I* B*

h n A

A
A

h n

F
I

h n

A
B

h n

F
B

h n

I

B

A

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

(negative OD) is associated with excitation of the chromophore ground state

(cf. Fig. 3 ). This is accompanied by the immediate appearance of positive

D

OD
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

D