study of green fluorescent protein

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Definition:

Green fluorescent protein (GFP is a natural protein which was first isolated from the jellyfish Aequorea victoria (2) that is after physical and chemical stimulation shows green fluorescent. The green emissions of this organism are under control of bioluminescent and chemilumienscent proteins, aequorin which is with blue emissions that require oxygen and calcium as cofactors and transfer energy to the GFP to represent green color.(3)

Relation to other bioluminescence:

A group of marine creatures both under anthozoan like Renilla or class Hydrozoa such as Obelia, Phialidium and Aequorea carry out green bioluminescent feature.(1)

At (395-470nm), GFP absorbs the maximum and minimum blue light respectively. Although purified GFP emits highest and lowest green light at (509 and 540nm). (7)

Structure:

GFP comprise 230 amino acids (7) and the crystal structure of the wild type GFP shows unique protein fold in cylindrical shape with 30 A° in diameter and a length of 40A. (4) This structure composed of eleven strand of ß sheet on the outside of the cylinder. In the middle of the β barrel, imidazolidinone chromophore move through symmetry axis with 60° angle which is responsible for the light(5). In addition, many short and deformed helical segments are in on one end of the structure. All these characteristics represent a name for protein, β can.( 2).

Physical characteristics of GFP:

According to results obtained from researches on A.victoria and R.reniformis, gfp genes are compact, acidic, globular molecules with monomer molecular weights of about 27kDa. They are stable which mean unless they exposed to denaturation, they remain dimeric.(2)

GFP stability:

One of the most important properties that have an advantage on gene expression is the high stability level of the protein. The main reason is the unique three dimensional structures. Chromophore (at the centre of B can) is highly protected and for that there is no effect of fluorescence agents such as molecular oxygen, acrylamide and halides on gfp fluorescence. The gfp purified from R.reniformis is the most stable in protein denaturing solution and has extreme PH stability.(2)

Effect of temperature:

The temperature at which the protein lost half of the fluorescence is 76ËšC for Aequorea and 70ËšC for Renilla.(2)

As a result of an increase in temperature, there is decay in uv circular dichroism signal at (205, 207 and 252.5nm) which explain there is loss of secondary and tertiary structure which in turn effect on the gfp fluorescence.(2)

Effect of proteases:

After 24 incubation of protein under moderate concentration of proteases, there is no effect on the fluorescence. GFP of Aeqorea and Renilla tolerate protease digestion such as trypsin, subtilisin and thermolysin in addition to its resistance to huge contamination of bacteria and fungi.(2)

Effect of organic solvents:

Treatment gfp with organic solvents result in a spectral shift (on the excitation spectrum only.

Acetonitrile is the most effective solvent that give these results of shifting. On the other hand, the exposure of Aequorea and Reniformis gfps to any solvents doesn't effect on neither excitation nor emission. But it results in intensity loss of fluorescence at solvent concentration > (60%v/v)(.2 )

Effects of detergents, fixation and preservatives:

There is no change in emission or excitation spectrum or intensity of any studied gfp types when trated with any detergents. Although, fixative such as glutaraldehyde formaldehyde and preservative compounds have no effects as well with gfp fluorescence. (2)

Uses:

As biosensors:

Biosensors based on gfp gave opportunity to monitor toxicity and pollution level in water or food samples. Some strain with modified gfp is more sensitive and detectable for genotoxin more than that of wild type gfp. However, E.coli strain with recA-gfp mutant 3 is a useful tool while responding to DNA damage caused by chemical agents such as nalidixic acid (NA) and N-methyl-N-nitro-N-nitrosoguanidine (NNNG).(IN DESKTOP)

Gene Expression:

To control gene expression and protein localization, gfp can be used as a critical marker. This protein doesn't need cofactors or added substrates but requires only illumining by blue light to be detected. (7)

Protein Labeling:

Cloning FP with the protein of interest at either C or N terminus is widely used today for protein tagging. (8)

PH-protein sensitivity: in desktop

GFP posses stable fluorescence at pH between 6-10. Spectral characteristics of chromophore (portion of the protein responsible of light emitting) depend on pH as a result of the ionization of the phenolic group (Tyr66). Phenolate form is preferred at higher pH and the absorbance is at 471nm while emission at 500nm. At low pH the phenol form shift the absorbance to 390nm. Absorbance shifting is due to conformational change of chromophore.

Application of ph:

Fusion tag:

GFP has been used to study various processes due to its stability over different pH environment. For example; it can be used to study movement of mitochondria through living cells by visualization and secretion, transportation of molecules by gfp fusion.

GFP variants:

A diversity of gfp variants have been generated in order to broad the range of GFP applications. For example; S65T GFP is a fascinating variant in having faster fluorophore formation, higher fluorescence intensity and altered excitation, emission spectra. (11)

Mutants of altered excitation and emission spectra are important in providing distinguishable markers to monitor multiple cellular events and to lighting the structure-function relationship of the unique protein.(10) Moreover, in order to study molecules, process in acidic or basic environments, specific variants are generated because of different PH sensitivities. (desktop)

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