Green Fluorescent Protein (GFP) has existed for more than one hundred and sixty million years in one species of jellyfish, Aequorea victoria. The protein is found in the photoorgans of Aequorea.
The discovery of GFP
The jellyfish Aequorea victoria is bioluminescent, i.e. it produces light with the help of
chemical reactions that provide the energy for photon emission and emits green light as first
described by Davenport and Nicol (1955). In 1960, Osamu Shimomura joined the laboratory
of Frank Johnson at Princeton to clarify the molecular mechanism of the bioluminescence of
Aequorea victoria. Shimomura came from Nagoya University, where he had completed
extensive work on the bioluminescence of the small ostracod Cypridina, together with Prof.
Y. Hirata. Aequorea victoria jellyfish were collected during the following summers in Friday
Harbor in the Puget Sound of Washington state on the coast of the Pacific Ocean
The active component of the Aequorea bioluminescence was identified as a protein, named
aequorin, emitting blue light in a Ca2+- dependent manner (Shimomura et al., 1962). That the
light emission of purified aequorin peaked in the blue part of the visible spectrum came as a
surprise, since the bioluminescence of Aequorea victoria is distinctly green.
GFP has a typical beta barrel structure, consisting of one β-sheet with alpha helix(s) containing the chromophore running through the center.Inward facing sidechains of the barrel induce specific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that lead to chromophore formation. This process of post-translational modification is referred to as maturation. The hydrogen bonding network and electron stacking interactions with these sidechains influence the color of wtGFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water.
The availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines.While most small fluorescent molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live cell fluorescence microscopy systems which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins. For example, GFP had been widely used in labelling the spermatozoa of various organisms for identification purposes as in Drosophila melanogaster, where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is spliced into the genome of the organism in the region of the DNA which codes for the target proteins, and which is controlled by the same regulatory sequence; that is the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e. dead) material. .
Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living organism. Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (Brainbow). Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of neuron membrane potential, tracking of AMPA receptors on cell membranes , viral entry and the infection of individual influenza viruses and lentiviral viruses,etc.
GFP in nature
The purpose of both bioluminescence and GFP fluorescence in jellyfish is unknown. GFP is co-expressed with aequorin in small granules around the rim of the jellyfish bell. The secondary excitation peak (480nm) of GFP does absorb some of the blue emission of aequorin, giving the bioluminescence a more green hue. The serine 65 residue of the GFP chromophore is responsible for the dual peaked excitation spectra of wild type GFP. It is conserved in all three GFP isoforms originally cloned by Prasher. Nearly all mutations of this residue consolidate the excitation spectra to a single peak at either 395nm or 480nm. The precise mechanism of this sensitivity is complex, but probably involves donation of a hydrogen from serine 65 to glutamate 222, which influences chromophore ionization. Since a single mutation can dramatically enhance the 480nm excitation peak, making GFP a much more efficient partner of aequorin, A. victoria appears to evolutionarily prefer the less-efficient, dual peaked excitation spectrum. Roger Tsien has speculated that varying hydrostatic pressure with depth may effect serine 65's ability to donate a hydrogen to the chromophore and shift the ratio of the two excitation peaks. Thus the jellyfish may change the color of its bioluminescence with depth. Unfortunately, a collapse in the population of jellyfish in Friday Harbor, where GFP was originally discovered, has hampered further study of the role of GFP in the jellyfish's natural environment.