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Role of Green Fluorescent Proteins and Their Variants in Development Of J Biosci Vol. 43, No. 4, September 2018, pp. 763–784 Ó Indian Academy of Sciences DOI: 10.1007/s12038-018-9783-0 Review Role of green fluorescent proteins and their variants in development of FRET-based sensors 1 1 2 3 1 NEHA SOLEJA ,OVAIS MANZOOR ,IMRAN KHAN ,ALTAF AHMAD and MOHD.MOHSIN * 1Department of Biosciences, Jamia Millia Islamia, New Delhi 110 025, India 2Faculty of Dentistry, Jamia Millia Islamia, New Delhi 110 025, India 3Department of Botany, Aligarh Muslim University, Aligarh, India *Corresponding author (Email, [email protected]) MS received 17 November 2017; accepted 11 May 2018; published online 3 August 2018 Since the last decade, a lot of advancement has been made to understand biological processes involving complex intra- cellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to ‘paint’ a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites. Keywords. FRET; green fluorescent proteins; metabolite; mutants; sensors 1. Introduction genetic tags has brought about advancement in the field of microscopy, particularly fluorescent microscopy (Kremers Atomic absorption spectroscopy, nuclear magnetic reso- et al. 2011;Cranfillet al. 2016). The strategy is to tag the nance, X-ray crystallography and other traditional electro- molecules with FPs and utilize fluorescence technology to chemical methods were used to obtain structural and explore biological systems. FPs can be used as probes to functional information about metabolites. Although these follow a molecular event, track the pathway of particular techniques provided an insight into the structural aspect, at metabolite and can also be incorporated into a sensor to thesametimetheysufferedfromchallengeslikelesssen- translate a cellular event into a biological signal (Van sitivity, poor resolution, invasive and non-selective nature Roessel and Brand 2002; Zhang et al. 2002). (Shimomura 2005; Mohsin et al. 2015). Progress in cellular FPs and novel imaging techniques together helped in biology required detailed, accurate and well-defined account revealing minute and crucial details of the dynamic beha- of the various metabolic processes and cellular pathways viour of a cell. Understanding biological systems involve inside the living cell with high-throughput screening of the elucidating details like quantification, cellular and sub-cel- various events. Later, sensors proved to be a promising tool lular compartmentalization, gene expression, molecular flux, to monitor, identify, quantify and track a biological event crowding, physiological activities, toxic effects and various within a living cell (Lippincott-Schwartz et al. 2001). Flu- intracellular/intercellular interactions (Tsien 1998; Ando orescent dyes have been used to fabricate sensors but their et al. 2002; Patterson and Lippincott-Schwartz 2002; Chu- toxic nature proved to be a loophole. Scientists started to dakov et al. 2003; Lippincott-Schwartz and Patterson 2003). look for other fluorescent alternatives and their search came FPs are being used extensively in imaging studies due to to an end with the discovery of fluorescent proteins (FPs). the following characteristics: The advantage of FPs is that they show bright fluorescence in theentireelectromagneticspectrum(Okumoto2010). They 1. They are not toxic to the living cells and therefore, can have simplified the task of imaging live cells, deep tissue and be expressed very efficiently. whole-body in vivo (Kremers et al. 2011). The use of FPs as 2. When co-expressed, FPs do not oligomerize. http://www.ias.ac.in/jbiosci 763 764 Neha Soleja et al. Figure 2. Calcium binding to aequorin releases blue light which was then absorbed by GFP to give green light. light to the bright green fluorescence (Patterson and Lippincott- Figure 1. Image of the jellyfish Aequorea victoria, which Schwartz 2002). Douglas Prasher isolated the GFP gene and produces the green fluorescent protein (GFP). later reported the 238 amino acid residues of the protein. Chalfie obtained the cloned GFP gene from Prasher and 3. They are photostable for extended time at physiological expressed it in Escherichia coli and Caenorhabditis elegans parameters. that glowed green on irradiation with UV light. He concluded that it can be used as a tracer molecule to follow the fate of any The 2008 Nobel Prize in chemistry was awarded to Osamu protein of interest. Roger Tsien explained the chemical reaction Shimomura, Martin Chalfie and Roger Y Tsien. They got of the synthesis of GFP which required oxygen. Tsien and his recognition for their efforts in making people realize how group created mutants with shorter life times compared to the important and beneficial are FPs for studying biological events. wild-type GFP which was brighter and also emitted a range of The end of the 20th century marked the discovery of green different colours (Chudakov et al. 2003). fluorescent protein (GFP) that called for a scientific revolution catching the interest of researchers. Shimomura was credited with the discovery of GFP. He was working on a project that 2. GFP as a protein tag required him to study a jellyfish, Aequorea victoria (figure 1) to understand the mechanism behind its bioluminescence. He The last decade of the 20th century has marked the begin- extracted a protein from the green glowing rings of the jellyfish ning of revolution in cellular biology. In 1990, various that was supposed to emit green light. But surprisingly it improvements were made by using mutagenesis to colour emitted blue light in the presence of calcium ions (figure 2). He shift the variants from blue to yellow. It is to be noted that named the protein as aequorin. This blue light was absorbed by GFP does not require any additional components other than another protein, GFP that was actually converting this blue oxygen to show green fluorescence confirming its ab O O Tyr 66 ) β-pleated sheet N Gly 67 N HO NH HO α-helix Ser 65 O Beta- (40 Barrel Figure 3. (a) Chromophore location within an a-helix inside the 11-b barrel of GFP. (b) The wtGFP chromophore, consisting of a cyclized tripeptide made of Ser65, Tyr66 and Gly67 (Cormack et al. 1996). Role of green fluorescent proteins in development of FRET-based sensors 765 O O O Tyr 66 Tyr 66 Cyclizaon and N Gly 67 Gly 67 H Dehydraon N O O NH N OH OH Nucleophilic aack of the amide HN HN of Glycine on the carbonyl Ser 65 O Ser 65 group of Serine OH HO O O O Imidazolinone ring formaon N NH HO OH HN OH Dehydrogenaon [O] O O O Tyr 66 N Gly 67 N OH HN Ser 65 O HO Mature GFP chromophore Figure 4. Sequential reactions resulting in the formation of a mature chromophore. autofluorescent characteristic (Prasher et al. 1992; Chalfie trans (Zimmer 2002). The two rings may be parallel or et al. 1994; Tsien 1998). perpendicular to each other. Due to the chromophore con- figuration and planarity, they are forced by the surrounding protein matrix to form hydrogen bonds and hydrophobic side 3. GFP as a chromophore chains which comprise the shape of an enclosing cavity. The importance of the closed cavity was discovered when Crystallography details reveal that GFP consists of an mutants at Tyr66 was analysed and shown to reduce 11-stranded b-can structure enclosing a central a-helix that brightness. contains the chromophore within itself (figure 3). The pri- There are two charged side chains that are conserved in all mary structure of the chromophore consists of a tripeptide, GFPs, namely Glu222 and Arg96 and these are present at the Ser-Tyr-Gly at positions 65–67 (Cody et al. 1993). Folding edges of the chromophore (Zimmer 2002). of the GFP molecule favours a cyclization reaction resulting in the formation of an amide bond between Ser and Gly. The presence of molecular oxygen breaks the a–b bond of Tyr66 5. Mechanism of fluorescence giving it a fully folded structure that enables the GFP molecule to show fluorescence (Ormo et al. 1996; Yang A photoisomerization model suggests that there are three et al. 1996; Zimmer 2002; Bishop et al. 2013) (figure 4). forms of the GFP chromophore: neutral, anionic and an intermediate state that explains the absorbance and emission spectra of wtGFP (wild type GFP) giving rise to two sepa- 4. Chromophore structure rate but coincident peaks. The neutral or the protonated form of the chromophore absorbs at 395 nm whereas the anionic The chromophore consists of two rings linked by a double or the deprotonated form absorbs at 475 nm. The emission bond. Due to restriction of groups around the double bond, peak observed at 510 nm on excitation of the chromophore the chromophore may exist in two configurations: cis and at 395 nm is caused by a process called excited-state proton 766 Neha Soleja et al. a b A Tyr 66 Gly 67 B Fluorescence Emission Intensity Ser 65 395 510 Wavelength (nm) c d A A* I B* (Intermediate state) 510 nm 5nm 395 nm 47 B - A B (Neutral or (Anionic or Protonated Deprotonated state) state) Figure 5. (a) Ball and stick demonstration of the GFP. Nitrogen atoms are blue and oxygen atoms are red in colour. (b) The absorbance spectrum of wild-type GFP, together with the chromophore forms (c) giving rise to the two peaks (Gu et al.
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