Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 188

COOH Amide A mide Fingerprint

0.1 Abs

1497

1147

1342

1580
1538

1316

0.004

H 2 O

1497

0.000

1343
1316

1149

1580
1539

D 2 O

-0.004

1800 1600 1400 1200 1000

Frequency (cm -1 )

Fig. 7 Reaction-induced difference FTIR spectroscopy characterises the photoconversion of
GFP. Reproduced with permission [ 20 ]. Figure legend: “Absolute FTIR spectrum of GFP in
water ( upper panel ) and FTIR difference spectra ( lower panel ) between neutral and anionic
species of GFP (i.e. GFP A GFP R )inH 2 O( middle ) and D 2 O( bottom ), showing the carboxyl,

amide I, amide II and fingerprint regions. An absolute spectrum (from 1,800 to 1,000 cm 1 ) was
obtained of the neutral form of GFP”

photoconversion and the associated strong red-shifted absorption [ 20 ]. Isotope
substitutions and density functional theory calculations on a model compound for
the GFP chromophore [ 41 ] suggest assignment of positive bands associated with
vibrations of the phenol group (1,497 and 1,582 cm 1 ), the imidazolidinone ring
(1,537 cm 1 ), and the bridging carbon (1,615 cm 1 ) of the anionic chromophore in
the room temperature IR difference spectrum [ 28 , 41 ].
A direct test of stable proton rearrangements such as proposed by Brejc et al.
(Fig. 4 ) by difference FTIR spectroscopy turned out negative, from direct measure-
ment of absorption changes belonging to COOH stretching vibrations in the
~1,720 cm 1 region [ 20 ]. A quantification of the GFP R -GFP A difference spectrum
showed that less than 0.1 carboxylate groups change protonation upon stable
photoconversion [ 20 ].
Insight into the mechanism of photoconversion of GFP came from the discovery
that covalent modification occurs upon UV or visible illumination. Specifically,
illuminated samples were shown to be reduced in mass by 44 Da, suggesting the loss
of a -CO 2 group [ 19 ] (Fig. 8 ). Structure solution of the purified photoproduct by
X-ray crystallography revealed the loss of the carboxylate group of the Glutamate
222 side chain [ 19 ] (Fig. 9 ). In the GFP R photoproduct, coordinate analysis suggests