Many distinctive properties of Green Fluorescent Protein (GFP) such as its ability to generate an internal fluorophore make it extra-ordinary. This review will set out to discuss not only the structural and biochemical properties, uses and interesting features of this unique protein but also the applications of new variants of GFP that have been developed.
GFP comprises of 238 amino acids, absorbs blue light (Amax 395 nm) to fluoresce green light (Imax 509 nm) (Miller et al, 2008). GFP has modified amino acids, not separately synthesized prosthetic group like the other fluorescent proteins and serve as energy-transfer acceptors, receiving energy from a Ca2+-activated photoprotein or a luciferase-oxyluciferin complex in a jelly fish Aequorea victoria (Prasher et al, 1992). Its importance in molecular biology was realized when wild type GFP was cloned and sequenced by Prasher et al in 1992. Although the literature covers a wide variety of uses and properties, this review will focus on some of the exclusive features of GFP.
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STRUCTURE-PROPERTY RELATIONSHIPS AND USES OF GFP
The crystal structure of recombinant wild-type A. victoria GFP (wtGFP) called a Î²-can was solved to a resolution of 1.9 Aâ° by Yang et al. Î²-can consists of a cylinder having 11 strands of Î²-sheets with an Î±-helix at each end with a chromophore in the middle. It has 2 excitation peaks, -at 395nm representing neutral chromophore GFP's and at 475nm for GFP's with anionic chromophores. It was seen that if more than the N-terminal Met or seven amino acids from C-terminal is deleted, there is loss of fluorescence (Yang et al, 1996).This maybe because the Î²-can structure cannot be formed even if a small part of the "can" is missing. According to Ormo. M et al(1996), the rigid encapsulation of the chromophore and tertiary structure of GFP is probably responsible for the small Stoke's shift, high quantum yield of fluorescence and inability of oxygen to appease the excited state.
Some important properties of GFP - resistance to pH, temperature and chemical-induced conformational changes; high extinction co-efficient; tendency for dimerization. The location of the chromophore within the compact single domain structure explains GFP's remarkable stability. GFP can also fuse either at the N or C terminal, because both termini of GFP are flexible on the surface of the beta can. (Chalfie, 1995).
WtGFP, at room temperature folds normally, but folding reduces at higher temperatures. This property is used in pulse-chase experiments where the fate of fluorescent protein designed at lower temperature is studied after reverting to normal temperature and simultaneous inhibition of new fluorescence. Most of the mutants are formed by Fluorescence Resonance Energy Transfer (FRET) wherein the emission spectrum of one fluorophore overlaps the absorption spectrum of another when they are in close proximity with each other. Various attempts of random and site-directed mutagenesis were made to create mutants, one of which used DNA shuffling in creating a triple mutant F99S, M153T, V163A which improved folding at 37â°C, decreased aggregation at higher concentrations and increased diffusibility of proteins within the cells. The rigid shell of GFP protects it from photo bleaching. (Tsien, 1998).
By studying the bond length variations in many models, a link between excitation energy and structural property of GFP was found which showed that distortion of chromophore accounts for protein-matrix effect on the excitation energy (Laino, 2004)
APPLICATIONS OF GFP AND GFP-VARIANTS
Due to its fascinating properties, GFP and its variants are widely used as markers for detection of gene expression in vivo by fluorescence microscopy, flow cytometry, macroscopic imaging techniques. For example, in Caenorhabditis elegans GFP serve as a reporter gene because the cuticle does not allow access of the substrates required for detecting other reporter genes (Tsien, 1998).GFP is used as an encodable pH indicator to study pH regulation in those cellular compartments where conventional pH dyes cannot probe. The main advantage of GFP is that it can respond to wider variety of cellular signals and biological activities as they are non-toxic.S202H GFP variant is used as an indicator to study peroxisomes, endosomes and Golgi networks. EGFP has been used to monitor pH variations in the cell cytoplasm but better green emitting variants, called "Ecliptic" and "Super ecliptic" were generated by mutagenesis which were excellent markers of cell exocytosis.E2GFP is used to study the cellular alkalinisation and the consequent acidification upon entry to the G1Phase in mitosis.E1GFP can monitor pH in real time during endocytosis. (Bizzarri, R, 2009)
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A gene encoding GFP fused with the gene encoding an endogenous protein results in a fusion protein that is expressed in the organism. One such GFP variant EGFP distributes itself homogenously within the cytosol of neurons. It gives us not only the detailed structure such as axons and dendritic spines, revealing distribution characteristics of proteins within neurons but also helps to record expression from a cell with a foreign gene (Malinow, 2010). Sensitive detection of live or fixed samples of Hepatitis C virus infected cells can also be done using an EGFP based reporter system (Jones et al, 2010).
Having 2 excitation peaks is the major deficiency of wtGFP. A variant S65T GFP mutant has only 1 excitation peak due to which there is a 6-fold increase in brightness, 4-fold increase in rate of oxidation of chromophore. This forms the basis behind enhanced GFP. (Zimmer. M, 2009)
Optical highlighter is a technology where research can be done in the future since we need genetically encoded photoactivable GFP's in super-resolution processes such as FPALM and STORM.GFP can find applications in brain mapping (Zimmer. M, 2009)
The development and understanding of GFP's have only begun and this review touches just the surface of all the utilities and versatility of GFP, its variants and fluorescent based techniques.