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Green Fluorescent Protein has become a invaluable tool in cell biology research due to its foster in advances in imaging technology in the living cell. This review is about the biochemical and structural properties of GFP and its mutants which give rise to Novel properties.
GFP is got from Aequorea Victoria a jelly fish which has a photoprotein Aequion which absorbs blue light (max 395 and a min absorbance of 475nm). This is transferred to the intact fluorescent protein, GFP where the efficiency of the light is increased and then it is emitted in the form of green light(507nm).The GFP has 238 amino acid and has a 11-Î² stranded barrel like a cylinder and the Î± chain runs around it and forms a lid on top called "Î² can ". Chromophore lies center inside the Î² can. GFP is activated either by the Lucifease-luciferin activated complex or the ca+ activated photoprotein. Changes in the covalent structure and the stereochemistry of the flurophore changes the obsorption and emission spectral of the wtGFP making it Novel for various studies. (Prasher, Eckenrode et al. 1992).
Fluorescence is due to the crystallisation of the tripeptides ser65, Tyr66, Gly67 and 1,2-dehydrogenation that leads to the formation of p-parahydroxybenzylideneimidazolelinone it is a aerobic reaction. If oxygen is not present fluororescence does not occur. The folding and the oxidation is a long process and it is not of biochemical interest. The protein is resistant to extreme conditions of pH upto 11 after which it loses its absorbance. It fluorescence upto 65C, resistance to protease enzyme and also chemical detergents like 1%SDS. GFPs are created with spectral variations of pH 7 but are not acid sensitive. This can be made advantages because if the light is quenched in a particular organal we can know the pH of the organel by knowing the sensitivity of the mutant.(Cubitt, Woollenweber et al. 1998; Tsien 1998).
WtGFP has a maximum absorption at 397 and a minimum at 475 due to the neutral phenols and the anionic phenolates and the emission is at 507. The extinction co-efficient of GFP was found to be 21-30mM-1 cm-1 and the quantum yield 0.72-0.85. when their product was calculated which gives the brightness it was found that the brightness is low compared to the free fluorescein . There are other problems of photoisomerization and photobleaching. Photoisomerization leads to decrease in the absorption of 395nm and increase of absorption in the 495nm this will lead to decrease in intensity of fluorescence at 395 illumination but fluorescence increases when illuminated with 495. Photoisomerization can be decreased by uncapping the fluorophore. Photobleaching leads to decrease in absorption of both the wavelength. Mutants have been created to decrease these problems one of such mutant is Photoactivation GFP (paGFP). Photoactivation is created where there is intense illumination and the fluorophore undergoes photoconservation and increases the minor peak absorption. This leads to three fold increase in the fluorescence. paGFP has increased optical enhancement under aerobic condition s making it suitable to mark protein and cell population.(Cubitt, Woollenweber et al. 1998; Patterson and Lippincott-Schwartz 2002) .
Once the cDNA for GFP was found different mutagenesis was done to the tripeptide and other aminoacid sequence to solve problems of aggregation, maturation problems at alleviated temperature, increase in intensity and energy transfer, extinction co-efficient, spectral variations and also photobleaching. Enhanced GFP is now created by shifting the absoption equilibrium toward the anionic side and it led to an increase the photostability, brightness, single wavelength emission and also the photostability. Mutation in Ser65T led to a good absorption of 470-490nm and emission of 510nm and there was a red-shift in the absorbance 489 and the maturation was faster, the extinction-coefficient six times higher and it is more resistant to photobleaching. Emission wavelength greater than red was not got due to the intrinc primary structure of GFP or the fluorescence property of GFP.(Remington 2000). Mutation in T66H and T145F also gives a variant fluorescent protein called Blue fluorescent protein( BFP). We can study the localization and also the expression of more than one proteins with introducing more spectral variant in the cell and then analysing them invivo with FRET for any protein protein interaction. The other mutant is the Photoactivation GFP (PA-GFP). Another interesting type is the Reversible switchable fluorescent protein (RSFP) here, due to irradiation of a specific light wavelength there may be fluorescence or no-fluorescence. This is because of the change in the cis-trans conformation of the chromophore in its protonation state (Stiel, Andresen et al. 2010) (Cubitt, Woollenweber et al. 1998; Patterson and Lippincott-Schwartz 2002; Zimmer 2009).
For its expression it requires a good promoter or enhancer. Modifications are made in the codon to increase expression or to make it Humanized GFP.
GFP due to its sensitivity and non-distructive monitoring of the gene can be used for as a reporter gene. GFPs can also be expressed in a heterologous system and does not require any exogenous substrates or cofactors to be expressed in the heterologous system. It can be used as a reporter gene by expressing with the endogenous protein and then the fluorescent is studied. It can be used as indicator by FRET technique. Here the molecule has got a strong surrounding where the outside molecule cannot interact with it. But now mutations are made to make GFP sensitive to the environment and there by detect the pH or the substrate present.
Here two fluorophore is introduced and each signal of donar and the recipient will interact with each other so that the emission does not take place. Once this linkage is cut on addition of some substrate there is a release of the emission.(Tsien 1998). By this we can study the molecules which cleave or the ligands which bind and change the protein.
Dicistronic expression can also be done where the GFP and the gene of interest is under the control of the same promoter.This reduces the screening process as the cell that expresses the GFP also expresses the protein of interest.(McLachlin, Cornetta et al. 1990)
Host pathogen effect
GFP is autocatalytic and can express continually in host cell. Its heterologous expression does not require any co-factor and is independent of the host cell. GFPs are also made humanized by introducing ribosomal binding site by codon modifications therefore it can introduced with the pathogen and the study of host-pathogen effect can be studied. It does not create toxicity or enter into the mammalian cell or the macrophages. It also does not involve in the cellular activity of the pathogen. This made to study Host-pathogen effect more effectively by analysing in the epifluoresence microscope. As the detection process used before was an Ab tagged with the fluorescent or to coat the pathogen with a fluorochrome and as the pathogen replicates its coating goes off. These problems are not in GFP cloned pathogen and also the pathogen can be quantitated with flow cytometry either alone or in association with macrophages. To make it humanized a ribosomal binding site is introduced. (Valdivia, Hromockyj et al. 1996; Valdivia and Ramakrishnan 2000).
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