Biology Reference
In-Depth Information
Keywords asFP595
Dronpa
Fluorescent proteins
GFP
Green fluorescent
protein
'Green-to-red' photoconversion
Kaede
Oxidative decarboxylation
Photoconversion
Photoisomerisation
Photoswitching
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
1.1 Molecular Structure Determination of GFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
1.2 The Fluorescence Photocycle of GFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
2 Photoconversion of A. victoria GFP ...................................................... 190
2.1 Discovery and UV/VIS Spectroscopic Investigations of Photoconversion of GFP . 190
2.2 Mechanism of Photoconversion of GFP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
2.3 Light-Induced Oxidative Decarboxylation in Other Fluorescent Proteins . . . . . . . . . . 200
3 Irreversible Green-to-Red Photoconversion in Related Fluorescent Proteins . . . . . . . . . . . . . 202
4 Light-Induced Maturation in Related Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
5 Reversible Photoconversion Reactions Resulting from Chromophore
Photoisomerisation in Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
6 General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
1
Introduction
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria was the first
fluorescent protein to be fully characterised spectroscopically and structurally. Key to
these successes was the molecular cloning of the gfp gene and its recombinant
expression in bacterial Escherichia coli cells [ 1 , 2 ]. Importantly, the light absorbing
p -hydroxybenzylidene-imidazolidinone chromophore forms autocatalytically. It is
this automatic maturation that allows the recombinant expression of the gfp gene
in vivo to fluorescently tag protein populations in living cells [ 3 ]. The crystal structure
of the S65T variant [ 4 ]andthewildtype[ 5 , 6 ] were subsequently solved. The
examination of the molecular structures enabled the creation of mutants with altered
spectroscopic properties, using both structure-based rational design and random
mutagenesis approaches, and it also allowed sample preparation for detailed bio-
physical studies [ 7 - 9 ]. The important utility of GFP and other fluorescent proteins
in fluorescent microscopy applications has revolutionised biological and medical
studies. They have also impacted many other fields, particularly the biophysics and
structural biology of light-induced reactions. With the number of available unique
fluorescent (and non-fluorescent) proteins that are reported in the literature at the time
of writing, there is a continuing research effort to chart and understand the diverse light
and dark reactions that occur in the native and mutant derivative proteins. While the
autocatalytic formation of a bright fluorescent gene product has already revolutionised
biological research that uses fluorescence microscopy techniques, the additional
photochemical characteristics of GFP and related fluorescent proteins have provided
even more advanced tools for molecular biologists. Biophysical research of light-
sensitive proteins has also made significant advances as a result. The discovery and
development of the GFP was awarded with the 2008 Nobel Prize in Chemistry to
Osamu Shimomura, Martin Chalfie and Roger Y. Tsien [ 10 ].
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