One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 3

2.1 Structure and Chromophore Formation

The 238 amino acid (27 kDa) long sequence of av GFP folds in a compact cylindrical
form called -sheet (Fig. 1 )[ 10 , 11 ].

Several X-ray structural studies support the notion that also all other FPs share the
b

b -barrel, its lateral wall being an 11-stranded b

-barrel fold. They can differ in quaternary structure, though most natural FPs are
tightly bound tetramers and some are dimers, a feature that initially hampered their
applications. However, mutagenesis studies were able in most cases to produce
viable monomeric variants of the parent proteins.
The

b -barrel is capped on both ends by short a -helical sections and traversed by

an -helix segment. This segment contains the chromophore, a 4-( p -hydroxyben-

zylidene) imidazolinone, originating from the posttranslational cyclization of
three consecutive amino acids at position 65-67, such as Ser, Tyr, and Gly. As
demonstrated by the fact that expression of av GFP gene in other organisms leads to
fluorescence, the posttranslational synthesis of the chromophore does not require
any jellyfish-specific enzyme [ 3 , 4 ]. It requires, however, exogenous oxygen, in the
absence of which GFP does not develop fluorescence [ 4 , 12 ].
Chromophore formation (Fig. 2 ) proceeds within the native fold (i.e., no chromo-
phore is formed under denaturing conditions). It first entails backbone cyclization at
the Ser65-Tyr66-Gly67 tripeptide through nucleophilic attack of the amide group of
Gly67 onto the carbonyl group of Ser65, promoted by Arg96, a conserved residue in
all natural FPs (see [ 13 ] and references therein). Molecular oxygen is required for the
subsequent oxidation reaction. One water molecule is also abstracted from
the structure, with most experimental studies supporting the cyclization-oxidation-
dehydration sequence of events [ 13 , 14 ].

a

Fig. 1 Tertiary structure of av GFP. The usual cartoon representation is used, where a -helices are
cylinders and b -sheets arrows . The chromophore is shown in a ball-and-sticks representation with
the standard coloration for atom elements (i.e., gray for carbon, blue for nitrogen, and red for
oxygen)