Biology Reference
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1.1 Molecular Structure Determination of GFP
An early breakthrough in GFP research was the successful crystallisation and
structure solution. Very close together in time the structures of the S65T mutant
[ 4 ], in which the anionic chromophore is stabilised, and the wild-type GFP [ 6 ], with
a predominantly neutral chromophore, were reported. Interestingly, the first crystals
of the native GFP isolated from A. victoria jellyfish were already reported in 1974
[ 11 ], and X-ray diffraction was recorded in 1988 [ 12 ]. However, it took many years
before its structure was finally solved, using multiple isomorphous replacement and
anomalous scattering [ 4 , 6 ] and molecular replacement [ 5 ]. The results showed that
GFP has a beta-barrel structure consisting of 11 beta strands with the light absorb-
ing p -hydroxybenzylidene-imidazolidinone chromophore fully embedded within
its core (Fig. 1 ). The availability of the structural coordinates started the rational
design of directed mutations that alter the spectroscopic properties.
GFP exists predominantly in the neutral phenol state, with the phenolic
p -hydroxybenzylidene-imidazolidinone chromophore protonated. The dominant
but partial stabilisation of this species GFP A (l max ¼
398 nm) at pH 8.0 results
from electrostatic repulsion by an acidic group, identified from X-ray crystallogra-
phy to be the carboxylate of Glu222 [ 5 , 6 ]. The thermal equilibrium properties of
proton transfer reactions include a plateau in the pH titration in the pH 7-10 region,
which is predicted from a four-state system that describes the interaction of the two
ionisable groups [ 13 ]. A comparison of the molecular interactions in the chromo-
phore region of the wild-type (Fig. 2a ) and the S65T mutant (Fig. 2b ) highlights the
stabilised neutral GFP A and anionic GFP B states, respectively. The stabilisation of
the neutral and anionic forms in these proteins is central to understanding the
spectroscopic details as well as the details of the photoconversion and the fluores-
cence reactions, which will be reviewed.
Fig. 1 (a) Crystal structure of the green fluorescent protein, drawn from 1GFL.pdb [ 6 ]. (a) A “side
view” of the secondary structure cartoon representation and (b) “end-on view”, showing the
p -hydroxybenzylidene-imidazolidinone chromophore buried inside the beta-barrel
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