Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 40

1 Introduction

The green fluorescent protein (GFP) was first discovered and isolated from the
jellyfish Aequorea victoria [ 1 , 2 ]. The subsequent sequencing, cloning and expression
of GFP revealed its remarkable utility in fluorescence imaging [ 3 ], a discovery which
has since lead to it becoming one of the major tools of cell biology [ 4 - 7 ]. Since the
discovery of wild-type GFP (avGFP), a substantial family of homologous proteins
has been discovered and characterised [ 8 ]. This family has in common the structural
features of an 11 stranded -helix

contains the chromophore, which is formed after protein folding by cyclisation and
oxidation reactions involving three adjacent amino acid residues - a serine, a tyrosine
and a glycine. The mechanism of chromophore formation has been extensively
reviewed [ 9 - 11 ]. Crucially, the final result is a chromophore with an extended pi
electron structure which is covalently bound to the protein backbone, absorbs in the
blue-green region of the spectrum and, in many cases, emits with a high fluorescence
quantum yield at green-red wavelengths. The chemical structure of the chromophore
was identified as p -hydroxybenzilideneimidazolinone (HBDI, Fig. 1 )[ 12 , 13 ]. The
high quantum yield, irreversible chromophore binding and the ease with which GFP
can be fused to a desired target protein make it (and its mutants) an ideal protein-
specific fluorescence label for live cell imaging [ 6 , 14 ].
The structure of the folded protein and the basic structure of the chromophore are
largely conserved throughout the ever widening GFP family. Despite this funda-
mental similarity, fluorescent proteins display an enormous range of photophysical
properties, which greatly extends their utility in imaging [ 15 , 16 ]. avGFP itself
exhibits two bands arising from the protonated and deprotonated form of the
phenolic hydroxyl group, and their relative proportion can be modified by both
pH and mutagenesis [ 17 - 19 ]. Excitation of the neutral form of avGFP results in an
excited state proton-transfer (ESPT) reaction, which is unique in biology [ 20 ]. The
energy of the electronic absorption and emission bands can be modified over a wide
range through mutagenesis [ 6 ]. Changes to the three amino acids that make up the
chromophore may lead to large spectral shifts, while more subtle but still substan-
tial shifts can be achieved through mutations in the surrounding residues [ 21 ]. The
variety of colours that result is critical in bioimaging applications. Multiple emis-
sion frequencies permit multicolour imaging allowing more than one protein to be
labelled in any given cell. When the energy levels of two GFP mutants are properly
aligned, fluorescence resonance energy transfer (FRET) may be observed between

b -barrel through which is threaded an a -helix. This a

O

CH 3

N

N

HO

Fig. 1 Structure of the
avGFP chromophore HBDI

CH 3