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Green Fluorescent Protein Avinash Bayya Varun Maturi Nikhileswar Reddy Mukkamala Aravindh Subhramani Introduction The Green fluorescent protein (GFP) was first isolated from the Jellyfish Aequorea victoria, widely found in North America. GFP has existed for more than one hundred and sixty million years in that species. GFP is a widely studied and highly exploited protein in the fields of biochemistry and cell biology. The Jellyfish also contains a bioluminescent protein called aequorin that emits blue light. When GFP is exposed to blue light, or ultraviolet light, it emits green fluorescent light. Therefore, when the protein aequorin binds with calcium and emits blue light, this blue light is in turn absorbed by GFP, which then emits bright green light. GFP has become an amazingly useful protein in scientific research because it allows us to examine the internal workings of the cell. It is easy to locate the GFP by just exposing it to ultraviolet light, since it then glows with a bright green fluorescent light. The trick lies in attaching the GFP to any object that you are interested in examining. For instance, you can attach the GFP with another protein and examine them through the microscope thanks to GFP’s fluorescence. Exploring the Structure The structure of GFP was solved in 1996 by Omro et al and Yang et al. Their structures were included in the PDB with the accession codes 1EMA and 1GFL, respectively. GFP consists of 238 amino acids with a molecular weight of 27 or 30 kD. The protein fold contains 11 anti-parallel beta strands and alpha helices. These strands and helices form a classical beta barrel structure. (Figure 1), enclosing the Ser-Tyr-Gly (chromophore) residues in order to protect them from solvent interactions. Thus, GFP is often referred to as “Light in the can”. Chromophore The chromophore is formed by p-hydroxybenzylideneimidazolinone formed from residues 65-67, which are Ser-Tyr-Gly of the native protein. The green shade in Figure 2 indicates the structure of the chromophore. The mechanism for the formation of the chromophore takes place in four steps; folding, cyclization, dehyration and oxidation. First the denatured GFP is folded to form the native conformation with a half time of 10 min. This is followed by the cyclization, where the imidazolinone is formed by nucleophilic interaction between the amide of Gly67 and the carbonyl of residue Ser65, and the dehydration. Then, the molecular oxygen dehydrogenates the bonds of the aromatic group of residue 66 in conjunction with imidazolinone (Figure 3). At this stage the chromophore is stable and can acquire visible absorbance and fluorescence. In the chromophore there is a special interaction between glycine and threonine creating a new bond, which in turn creates an unusual five-membered ring (Figure 4). Figure 4. Chromophore illustrated in SwissPDBViewer. GFP, when viewed at high resolution, offers great explanation between the protein structure and spectroscopic functions. The chromophore is situated in the middle of the GFP, totally protected from the environment. This shielding is essential for the fluorescence. When the chromophore absorbs a photon, jostling water molecule would take away the energy of the chromophore. But inside the protein, the chromophore is protected by releasing a less energetic photon of light, instead of releasing energy. Some important polar groups buried beside the chromophore are Gln69, Arg96, His148, Thr203, Ser205 and Glu222. Also, GFP’s dimeric form is highly influenced by the compulsory conditions for its expression. Changes in GFP results in the release of new colors Substitutions of the amino acids in the chromophore region results in change of GFP color when exposed to blue or ultraviolet light. There are three GFP variants; blue fluorescent protein (BFP), yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). The amino acid substitutions in the GFP variants are shown below (Table 1). Table 1. Amino acid substitutions in the GFP variants. Amino acids Color GFP Green 65 66 67 68 69 72 145 146 153 163 203 wtGFP Phe Ser Tyr Gly Val Gln Ser Tyr Asn Met Val Thr Green EGFP Leu Thr Blue EBFP Leu Thr Yellow EYFP Phe Gly Cyan Leu Thr ECFP 64 His Phe Leu Trp Ala Tyr Ile Thr Ala The above table represents the position of the amino acid in various GFP variants. These GFP variants are more enhanced than the wild type GFP and the result is that more fluorescence can be seen in mammalians. These variants can be used as reporter genes in order to learn about protein interactions, timing of cell cycle events, and much more. GFP as a biosensor GFP is used within various fields of biology like molecular biology, neuroscience and cell biology. It can be used as a reporter gene, cell marker, fusion tag, and for quantitative monitoring of gene expression. GFP’s fluorescence characteristic can also be used in methods designed for sensing various levels of ions or pH. As mentioned above, there have been many codon alterations performed in GFP in order to make the expression effective in mammalians. As an application in genetic engineering, fluorescent green rabbits, rats, mice, frogs, flies, worms, and other living organisms have been produced. Below, the use of GFP as a biosensor for measuring the concentration of lead in aquatic environments will be described. Here, the GFP gene is under the control of the lead resistance regulatory gene (PbrR) and its operator promoter (PbrO/P), originating from the plasmid pMOL30 of Ralstonia metallidurans. The regulatory gene and its promotor were isolated by PCR using the PbrR/O/P forward and reverse primers carrying Nhe1 and Age1 sites, respectively. The amplified PCR product was cloned into the pDB402 plasmid containing the gfp gene. The entire PbrR/O/P/GFP was excised using Nhe1 and EcoRV and inserted into the pBR322 vector, and transformed into E. coli DH5α cells. A correctly cloned and confirmed sequence was named the Pbr-GFP biosensor (Figure 5). The Pbr–GFP biosensor was grown in LB broth with 100 µg/ml ampicillin at 37OC, and streaked on to LB-amp plates with various concentrations of lead ions (Pb2+). The plates were incubated at 37OC for 16h. The fluorescence was then directly measured using the Zeiss florescence stereozoom microscope at 25× at an excitation range of 450-490 nm and an emission range of 500-550 nm. The expression of GFP was evaluated at the following Pb2+ concentrations: 50, 100, 150, 200, 250, 300, 350, and 400 µM. The fluorescence showed a steady increase from 50 µM and upwards, peaked at 250 µM, and then again decreased. The fluorescence was also highest after 12 h. The following formula (based on the 12 h measurements) was derived using multiple regressions, and it can be used in order to estimate the Pb2+ concentration in an unknown water sample with 95% accuracy. C = (1629.02458 + (-0.00010809×D) + (0.00000502×F) Where, C: Concentration of Pb2+ in the sample D: Cell density F: GFP fluorescence in terms of RLU (Relative Light Unit) 1629.02458, -0.00010809 and 0.00000502 are constants. Discussion GFP can be used as a reporter gene; it shows high sensitivity, it is non-toxic, time efficient and cost effective when compared to the other classical reporter genes like lacZ and GUS. Many spectral variants of GFP are now available, and therefore it is possible to label different proteins, in different colors, inside the same cell. GFP can also be used as an active tool in finding the dynamics of chromosomes in vivo. It can be used in the studies of active bacterial chromosome segregations, yeast mitosis, and centromere dynamics, by tagging it to specific chromosomes. As a last example, GFP can be used as a biosensor for detecting the lead ion concentration in water. It is easy to use this technique to estimate the concentration of any simple substance in the water that can be grown in media. References • Virapong Prachayasittikul, Chartchalerm Isarankura-Na-Ayudhya, Yaneenart Suwanwong, Srisurang Tantimavanich., Construction of chimeric antibody binding green fluorescent protein for clinical application, EXCLI Journal 2005;4:91-104. Tapas Chakraborty, P. Gireesh Babu, Absar Alam and Aparna Chaudhari., GFP expressing bacterial biosensor to measure lead contamination in aquatic environment, CURRENT SCIENCE, VOL. 94, NO. 6, 25 MARCH 2008. Roger Y. Tsien., Annu. Rev. Biochem. 1998. 67:509–44 • http://www.cryst.bbk.ac.uk/PPS2/projects/jonda/intro.htm • http://www.ebbep.org/docs/pglo/gfpstructure.pdf • http://www.clontech.com/images/pt/PT2040-1.pdf • http://www.conncoll.edu/ccacad/zimmer/GFP-ww/structure.html • http://www.cryst.bbk.ac.uk/PPS2/projects/jonda/chromoph.htm • • http://www.pdb.org/pdb/explore/images.do?structureId=1EMA http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm • •