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 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 -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, , 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 (GFP). later reported the 238 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 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) 1 nm 510 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. 2011) (d) Photoisomerization mechanism of GFP.

resulting from excitation at 395 and 475 nm is quite similar because in the excited state, the phenolic oxygen of Tyr66 is comparatively more acidic than in the ground state. Thus, on excited-state proton transfer identical anionic excited states are generated (figure 5) (Remington 2011). The emission of fluorescence from the anionic chro- mophore is due to the falling of an electron from the singlet excited state back to the ground state. Protonation of the Fluorescence Intensity chromophore results in quenching of the fluorescence. When 450 455 485 535 600 the GFP chromophore was compared with other model Wavelength (nm) compounds, emission of the excited state of the protonated chromophore was observed in the green region rather than in Figure 6. Fluorescence excitation (blue) and emission (green) spectra of native GFP from Aequorea victoria. blue (Ward et al. 1982). Ultrafast fluorescence upconversion spectroscopy further revealed that when 397 nm light is used for excitation, expected blue emission at 460 nm is observed transfer in which the proton is transferred from the chro- (figure 6), but this blue emission decays with biphasic time mophore’s tyrosyl group to a proton acceptor, resulting in a constants. This decay in blue gives rise to green emission at deprotonated chromophore form. The two emission peaks 510 nm (Chattoraj et al. 1996). Role of green fluorescent proteins in development of FRET-based sensors 767

Blue Green Yellow Red Far-red Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Proteins Proteins Proteins Proteins Proteins

Cyan Orange Fluorescent Fluorescent Proteins Proteins

Figure 7. Variants of GFP spans the entire electromagnetic spectrum.

6. Variants of GFP minimum photobleaching (Cody et al. 1993). The increased stability and brightness of EGFP enables monitoring of Introduction of FPs such as GFP aroused excitement among metabolites and permitting the quantification of molecules researchers and they started looking for ways of ‘painting’ visualized within cells (Ormo et al. 1996; Yang et al. 1996). the living cells with a wide array of different colours, that Emerald, coral reef derived ZsGreen and T-sapphire are too at the same time. Despite its ground-breaking discovery, some much improved variants of the green palette. Emerald GFP was reported to have certain limitations such as pH and has four more point in it other than the two (S65 T chloride ion sensitivity, less photostability and shows poor and F64 L in EGFP) which improves its brightness, folding expression at 37°C. Mutations in GFP yielded variants with and maturation at 37°C but it photobleaches readily. improved characteristics in the protein such as rapid and These variants mature significantly faster than EGFP and better folding, brightness, photostability and easy expres- show much greater resistance to changes in pH which makes sion. Efforts were made to increase the quantum yield, molar them an ideal fusion tags for targeted expression of proteins extinction coefficient, photostability and overall brightness in acidic environments. Despite better optical properties and of the variants. Mutagenesis in wild-type GFP has resulted in folding kinetics of these proteins, they were reported to show new FPs covering blue to the yellow range of the spectrum. poor photostability, still less tolerant to pH changes and Anthozoan species has revealed many other FPs that emit in resulted in formation of aggregates at higher temperatures. the red and far-red regions of the spectrum. Most of the FPs Further developments in GFP provided variants with are known to exist in oligomeric forms. Single amino acid different absorbance and emission spectra, allowing the substitution, Ser to Thr (S65 T) amplified the rate of fluo- simultaneous visualization of distinct GFP variants in a cell. rophore formation (Heim et al. 1995) and several other Other mutations have produced blue FPs (BFPs) and cyan mutations helped in the folding of the molecule at 37°C FPs (CFPs) (figure 7). BFP has a substitution of histidine at (Tsien 1998). The protein expressions of the mutated forms position 66 in place of tyrosine and shows absorbance at 384 of the GFP variants were improved (Tsien 1998), and the nm with an emission peak at 448 nm. BFP variants were dimerization of the GFP at high concentrations was over- majorly used in intracellular imaging (Heim and Tsien 1996; come by the mutations Ala206 to Lys206, Leu221 to Lys221 Rizzuto et al. 1996) and fluorescence resonance energy or Phe223 to Arg223 (Zacharias et al. 2002). The major and transfer (FRET) experiments. Blue variants were faint (Heim minor absorbance peaks of wild-type GFP were converted to and Tsien 1996; Cubitt et al. 1999) and used to have pho- a single peak at 489 nm by substituting serine at position 65 tobleaching easily and readily, therefore other multicolour with a threonine, alanine, glycine, cysteine or leucine (De- pairs were created (Ellenberg et al. 1998). EBFP was one of lagrave et al. 1995; Heim et al. 1995). This forms the the first blue variant developed but remains an unpopular brighter molecules which can fluorescence better than the choice because of poor photostability and limited brightness wild-type molecules (Cubitt et al. 1999). These improved (Shaner et al. 2007). BFP and EGFP were considered as a characteristics are combined in the GFP variant, enhanced popular choice to be used as FRET pairs in multicolour GFP (EGFP), improve the GFP expression in mammalian imaging as the emission spectra of BFP overlaps to a great cells (Tsien 1998), hence the name started as enhanced. The extent with the excitation spectrum of the donor. Also, BFP S65 T variant is extremely bright and stable (Heim et al. has a distinguishable emission profile from EGFP, enabling 1995), and Phe64 to Leu64 (GFP2) enhance the their utility in tagging of targeted proteins. However, at the temperature sensitivity of GFPs (Cormack et al. 1996). same time, BFP limits itself as it shows excessive photo- Proteins tagged with EGFP can enable the visualization of bleaching, limited brightness, phototoxicity and autofluo- cells even with low intensity for a longer time with rescent issues. BFPs are usually excited by the wavelength 768 Neha Soleja et al. that falls in the spectral region which hampers multicolour imaging and localization studies as it shows cells and tissues by inducing toxicity and effecting phyto- considerably greater photostability. ECFP and yellow FP physical applications. Other common and improved FPs (YFP) derivatives are by far the most commonly used FRET lying in the blue spectrum are Azurite, SBFP2, EBFP2 and pairs for constructing molecular sensors. Certain dimeriza- mTagBFP. tion problems are also associated with these biosensors, CFP has spectral characteristics intermediate between thereby giving rise to two monomeric variants (mCFP and BFP and EGFP due to a Tyr66 to Trp66 substitution. It is mYFP) by replacing the hydrophobic residues at the dimer brighter (Heim and Tsien 1996) and is more photo- interface with positively charged amino acids (Oleynch et al. stable compared to BFP. ECFP is only 30% bright compared 2007). AmCyan1 (more photostable and has improved to its green counterpart. ECFP is preferred over EBFP in brightness) was derived from coral reef, Anemonia majano

O O R1 O His 66 O R1 Trp 66 N HN N N N

NH N

N NH HO R H 2 R2 HO O O EBFP ECFP

O O O O R1

R1 N N HN N N HO NH NH

HO HO R2 R Thr 65 2 Trp 66 O O EGFP mHoneyDew

O O O O R Tyr 67 1 Tyr 67 R1 N N N HO Thr 66 N N HO

N O H2N(H2C)2 R 2 Gln 66 R2 mOrange O DsRed

Figure 8. Some naturally occurring FP chromophores, coloured roughly in accordance with fluorescence emission. Role of green fluorescent proteins in development of FRET-based sensors 769

Table 1. Phytophysical properties of some of the most popular and useful fluorescent proteins (FPs)

Molar Emission extinction Excitation peak coefficient Quantum Relative Protein peak (nm) (nm) (M-1 cm-1) yield Structure brightness (% of EGFP)

Blue EBFP 383 445 29,000 0.31 Weak 27 Fluorescent dimer Proteins EBFP2 383 448 32,000 0.56 Weak 53 dimer Azurite 384 450 26,200 0.55 Weak 43 dimer mTagBFP 399 456 52,000 0.63 Monomer 98 Sapphire 399 511 29,000 0.64 Weak 55 dimer T-Sapphire 399 511 44,000 0.6 Weak 79 dimer

Cyan ECFP 439 476 32,500 0.4 Weak 39 Fluorescent dimer Proteins mCFP 433 475 32,500 0.4 Monomer 39 Cerulean 433 475 43,000 0.62 Weak 79 dimer mCerulean 433 475 43,000 0.62 Monomer 79 CyPet 435 477 35,000 0.51 Weak 53 dimer AmCyan1 458 489 44,000 0.24 Tetramer 31 Midori-Ishi 472 495 27,300 0.9 Dimer 73 Cyan TagCFP 458 480 37,000 0.57 Monomer 63 mTFP1 462 492 64,000 0.85 Monomer 162 mTurquoise 434 474 30,000 0.84 Monomer 75 mTurquoise2 434 474 30,000 0.93 Monomer 83

Green GFP2/EGFP 484 507 56,000 0.6 Weak 100 Fluorescent dimer Proteins Emerald 487 509 57,500 0.68 Weak 116 dimer Superfolder GFP 485 510 83,300 0.65 Weak 160 (sfGFP) dimer Azami Green 492 505 55,000 0.74 Monomer 121 mWasabi 493 509 70,000 0.8 Monomer 167 TagGFP 482 505 58,200 0.59 Weak 110 dimer TurboGFP 482 502 70,000 0.53 Dimer 102 AcGFP 480 505 50,000 0.55 Weak 82 dimer ZsGreen 493 505 43,000 0.91 Tetramer 117 Clover 505 515 111,000 0.76 Monomer 251 mUKG 483 449 60,000 0.72 Monomer 129 mNeonGreen 506 517 116,000 0.8 Monomer 276

Yellow EYFP 514 527 83,400 0.61 Weak 151 Fluorescent dimer Proteins Venus 515 528 92,200 0.57 Weak 156 dimer Topaz 514 527 94,500 0.6 Weak 169 dimer SYFP2 515 527 101,000 0.68 Monomer 204 mCitrine 516 529 77,000 0.76 Monomer 174 YPet 517 530 104,000 0.77 Weak 238 dimer TagYFP 508 524 62,000 0.6 Monomer 118 PhiYFP 525 537 124,000 0.39 Weak 144 dimer ZsYellow1 529 539 20,200 0.42 Tetramer 25 mBanana 540 553 6,000 0.7 Monomer 13 770 Neha Soleja et al. Table 1. (continued)

Molar extinction Relative Excitation Emission coefficient Quantum brightness (% of Protein peak (nm) peak (nm) (M-1 cm-1) yield Structure EGFP) Orange Kusabira 548 559 51,600 0.6 Monomer 92 Fluorescent Orange Proteins Kusabira 551 565 63,800 0.62 Monomer 118 Orange2 mKOk 551 563 105,000 0.61 Monomer 178 dTomato 554 581 69,000 0.69 Dimer 142 TagRFP 555 584 100,000 0.48 Monomer 142 TagRFP-T 555 584 81,000 0.41 Monomer 99 mOrange 548 563 71,000 0.69 Monomer 116 mOrange2 549 565 58,000 0.6 Monomer 104 mTangerine 568 585 38,000 0.3 Monomer 34 Red DsRed 558 583 75,000 0.79 Tetramer 176 Fluorescent DsRed2 563 582 43,800 0.55 Tetramer 72 Proteins DsRed- 556 586 35,000 0.1 Monomer 10 Monomer HcRed1 588 618 20,000 0.015 Dimer 1 mRFP1 584 607 50,000 0.25 Monomer 37 mCherry 587 610 72,000 0.22 Monomer 47 mRuby 558 605 112,000 0.35 Monomer 117 mApple 568 592 75,000 0.49 Monomer 109 mStrawberry 574 596 90,000 0.29 Monomer 78 mRaspberry 598 625 86,000 0.15 Monomer 38 tdTomato 554 581 138,000 0.69 Monomer 283 Far-red mKate2 588 633 62,500 0.4 Monomer 74 Fluorescent mPlum 590 649 41,000 0.1 Monomer 12 Proteins mNeptune 600 650 67,000 0.2 Monomer 40 HcRed- 590 637 160,000 0.04 Monomer 19 Tandem NirFP 605 670 15,700 0.9 Dimer 42

The relative brightness values were computed as: product of the molar extinction coefficient and quantum yield divided by the value for EGFP (Shaner et al. 2007; Piston et al. 2012).

(Piston et al. 2012). This variant is optimized for codon brightness, photostability and halide sensitivity. Variants of usage in humans. Cerulean and mCerulean are two-fold YFP suffer from their sensitivity towards low pH hence brighter than ECFP. CyPet, TagCFP and mTFP1 are some makes them an unpopular choice for tagging proteins that other cyan variants developed so far. Y66W mutant, mTFP1 are targeted to acidic environments. CyPet–YPet can become (teal) is slightly red-shifted and has high-quantum yield one of the most suitable FRET pairs for localization studies, compared to mECFP and mCerulean (Shaner et al. 2007). FP assays and multicolour imaging applications (Oleynch YFP is designed to have red-shift absorbance and emis- et al. 2007). ZsYellow1, another variant derived from Indian sion spectra with respect to EGFP and other fluorescent and Pacific coral reefs and YPet do not photobleach easily variants. When excited at 514 nm, YFP is much brighter and can be used for expression in acidic environments than EGFP, but is more sensitive to low pH and high-halide (Shaner et al. 2007). concentrations. Attempts have been made to improve Efforts were made to further red-shift the FP spectra. brightness, maturation rate and expression level at 37°Cin mKO, mOrange, tdTomato, TurboRFP and TagRFP, derived several variants of YFP. EYFP is another yellow variant with from sea anemone Entacmaea quadricolor, emit wavelength high pKa value and less sensitivity to halide ions. Citrine and in the orange region of the spectrum and are classified as Venus are brightest and fastest maturing among other vari- orange fluorescent proteins (Shaner et al. 2007). High pho- ants and also resistant to halide ions, and comparatively tostability, less pH and acidic sensitivity, rapid folding and more photostable (Piston et al. 2012). The monomeric maturation rate make these variants promising acceptor (mCitrine and mVenus) forms of these variants are also molecules in FRET pairs. commonly used. As far as expression in mammalian cells is DsRed discovered in anthozoan corals (Discosoma stri- concerned, Topaz and super YFP are efficient in terms of ata). The red-shift emission is due to oxidation of the protein Role of green fluorescent proteins in development of FRET-based sensors 771

OH abO c O O Trans form Tyr 66 Tyr 63 Gly 67 Tyr 63 O N Gly 64 N O N HO N O NH O Gly 64 Ser 65 N O N OH HN Quiescent state NH Fluorescent state NH (dark) Quiescent state Glu 222 (Green) HS O (dark) O His 62 N Cys 62

Decarboxylaon of Glu222 results Cleavage of the bond between Cis-trans isomerizaon in photoacvaon of PA-GFP from the amide nitrogen and α-carbon results in photoswitching quiescent (dark) state to an atoms in His62 residue of the chromophore 488nm anionic state (eming green 405 nm photoconverts the chromophore 405 nm from dark state to fluorescence) from green to red. fluorescent state.

Cis form O O O Tyr 63 O O Tyr 66 Tyr 63 O N Gly 67 N Gly 64 N N N Gly 64 HO O N Ser 65 NH O NH O OH NH HS NH2 O Fluorescent state Fluorescent state Fluorescent state Cys 62 (Green) His 62 O Glu 222 (Red) N (Green)

Figure 9. Mechanisms of optical highlighters: (a) photoactivation; (b) photoconversion and (c) photoswitching. backbone, which had extended the chromophore conjugation mApple and mStrawberry (75%) are red members of the by one double bond compared to GFP but with a little Fruit family with enhanced brightness over EGFP. mKate success (Shaner et al. 2007; Piston et al. 2012). DsRed is (extremely photostable, brighter) and mPlum emits in the considered to be toxic for tagging proteins because of slow far-red region and can be promising fluorescent probes for maturation, ‘green’ state and has tetrameric characteristics. FRET experiments (Shaner et al. 2007; Piston et al. 2012) DsRed2 (an improved variant) retains its obligate tetrameric (figure 8; table 1). form but shows increased maturation rates owing to muta- tions at the amino terminus of the protein, making it a better fluorescent tag while conducting multi-imaging experiments. 6.1 FPs as optical highlighters Red-Star is another brighter and photostable protein of the red palette and is a yeast-optimized mutant. HcRed extracted Complex photophysical properties of FPs have led to the from Heteractis crispa show less brightness with respect to generation of a new class of chromophores called optical its counterparts and therefore considered less applicable for highlighters. There are three classes of optical highlighters its use in live cell imaging (Piston et al. 2012). Shaner et al. (Shaner et al. 2007; Piston et al. 2012): (2004) further extended this effort by coming up with ‘mFruits’ collection of bright, photostable monomeric pro- • Photoactivatable (‘off’ to ‘on’ state): upon illumination teins ranging from yellow to deep red in emission. Mono- by ultraviolet light, these FPs can be activated to initiate meric forms of FPs are better options for FRET experiments bright fluorescence emission from a quiescent/dark or since dimerization may often give complex data. The ground state; for example: PA-GFP, PAmCherry and improved monomeric versions have greater quantum yields, PAmKate. increased photostability and higher extinction coefficients. • Photoconvertible (colour change): these FPs show Monomeric RFP (mRFP) photobleaches quickly than its transition from one fluorescence emission bandwidth to counterparts, mGFP and mYFP. mCherry (50%), mRuby, another; for example: Dendra2, Kate and EosFP. 772 Neha Soleja et al. • Photoswitchable: illumination at a particular wavelength molecules must reside within 10 nm of each other (fig- can alternatively turn these FPs ‘on’ (i.e. shows fluores- ure 10b) and (iii) emission dipole of the donor and the cence emission) to ‘off’ (i.e. quench fluorescence) and absorption dipole of the acceptor must not be perpendicular then back to the ‘on’ state; for example: Dronpa, to each other (figure 10c). Kindling FP and Dreiklang. Studies of reversibly Transfer of energy depends on the inverse sixth power of photoswitchable GFPs such as Dronpa and mTFP0.7 fluorophore distance. Thus, when the fluorophores come revealed that in the ‘resting’ state these are brightly closer, there is a decrease in the donor chromophore emis- fluorescent, but when illuminated, they rapidly photo- sion while an increase in the emission of the acceptor bleach and become non-fluorescent (Remington 2011) chromophore (figure 11). The Fo¨rster distance (Ro)is (figure 9, table 2). defined as the distance where the transfer of energy is 50%. R is a function of the spectral overlap between donor GFP has a number of marvellous properties that enable its o emission and acceptor excitation spectra, the quantum yield use for in vivo imaging. Following features have made GFP of the donor in the absence of the acceptor and the relative the most useful fluorescent protein: orientation of donor and acceptor chromophore transition 1. The GFP chromophore requires no substrates or cofac- dipoles (Fo¨rster 1965). The detection and quantitation of tors, therefore used to examine living cells with minimal FRET can certainly be accomplished in a number of different invasive. ways. FRET can cause decrease in fluorescence of the donor 2. Subcellular localization of any protein can be detected molecule as well as an increase in fluorescence of the by fusing GFP to that protein without loss of acceptor; therefore, a ratiometric determination of the two fluorescence. signals can be made (Jares-Erijman and Jovin 2003). 3. Since GFP is a protein, cDNA can be introduced to make a cell fluorescent and is inheritable. 9. Role of GFP in FRET It is possible to mutate the GFP and create the variants with improved features (Chalfie and Kain 1998; Conn 1999). One of the most significant uses of GFP-like proteins is The use of GFP is a vigorous technology that has enabled constructing genetically encoded fluorescent sensors for researchers to study dynamic biochemical events within various analytes and to study the protein activities. It is a tool living cells. A method for detecting molecular interactions for monitoring biological activities in time and space involves FRET between two GFP variants. through the use of illumination (Remington 2011). Such proteins have been exploited to optically control cellular processes both ex vivo and in vivo. Genetically encoded 7. Fluorescence resonance energy transfer fluorescent dyes, such as GFP and other related molecules blue, cyan, yellow and red have provided the ability to The theoretical analysis was well conducted by Theodor perform FRET in vitro and particularly in living cells (Tsien Fo¨rster in 1948. It is a non-radiative transfer in which a 1998). These proteins form the FRET pairs. donor group excited by a photon returns to the lowest excited singlet state. If the acceptor group is closer, then the energy released when the electron returns to the ground state 9.1 Opening the gate to the nanoworld (S0) is used to excite the acceptor. This non-radiative process of energy transfer may be referred to as ‘resonance’. After The technological advancements in FPs along with sophis- excitation, if the other quenching states do not exist, the ticated robust imaging techniques contributed to the success excited acceptor emits a photon and returns to the ground of FRET-based investigation of cellular processes of bio- state (Fo¨rster 1965). The origin of fluorescence lies in logical importance. Towards the end of the 19th century, inhibition of internal motion-driven non-radiative process optical microscopy was speculated to suffer from a setback due to the rigid protein matrix. defined as Abbe’s limit, i.e. no two elements of a structure can achieve a better resolution than half of its wavelength. Surpassing the limitations of light microscopy, FRET along 8. Basic requirements of FRET with novel microscopic and spectroscopic techniques has enabled multi-event imaging possible explaining cellular For resonance energy transfer to occur, three specific con- interactions of macromolecules within their native environ- ditions should meet: (i) emission spectrum of the donor ment (Mo¨ckl et al. 2014). fluorophore and absorbance spectrum of the acceptor Dimerization studies introduced digitized video FRET molecules must overlap; the overlap is referred to as the (DVFRET) microscopy, but out-of-focus emission signals spectral overlap integral (figure 10a); (ii) donor and acceptor from above and below the focal plane limited the contrast of Table 2. Fluorescent proteins as optical highlighters: (a) photoactivation; (b) photoconversion and (c) photoswitching sensors FRET-based of development in proteins fluorescent green of Role

Excitation Emission Molar extinction Quantum Relative brightness Protein peak (nm) peak (nm) coefficient (M-1cm-1) yield Structure (% of EGFP) Example and Reference Photoactivation PA-GFP 504 517 17,400 0.79 Monomer 41 (G)

(Patterson and Lippincott-Schwartz 2002; Piston et al. 2012) Photoconversion (G) 508 518 98,800 0.88 Tetramer 259 Dendra2 490 507 45,000 0.5 Monomer 67 (G)

(Sattarzadeh et al. 2015; Piston et al. 2012) Photoswitching PS-CFP 402 468 34,000 0.16 Monomer 16 (C) PS- 490 511 27,000 0.19 Monomer 15 CFP(G) Dronpa 503 518 95,000 0.85 Monomer 240 (G) (Olenych et al. 2007; Eisenstein 2005) Kindling 580 600 59,000 0.07 Tetramer 12 (KFP1) 773 774 Neha Soleja et al. (a) Spectral Overlap between donor’s emission and acceptor’s absorbance spectrum

CFP YFP overlap

(b) Distance between the donor and acceptor fluorophores should be less than 10nm

485 435 nm nm 535 nm

CFP YFP

> 10nm

(c) Parallel orientation of the two fluorophores

FRET No FRET 435 nm 535 nm

CFP YFP CFP YFP

Figure 10. Conditions for FRET: (a) spectral overlap; (b) distance \10 nm and (c) correct orientation.

the images produced. It led to the development of confocal thus making FRET investigate cellular events in non-inva- FRET (CFRET) microscopy for single-molecule FRET (sm- sive and non-intrusive manner. Fluorescence correlation FRET) as it was able to overcome the problem of out-of- spectroscopy (FCS) is one of the earliest emerging tech- focus signals but suffered from two major limitations, pho- niques that uses autocorrelation to observe fluorescent tag- tobleaching and photodamage of the fluorescent molecules. ged molecular events in intact living cells. In contrast, Two-photon FRET (2p-FRET) microscopy was a better fluorescence cross-correlation spectroscopy (FCCS) is more improvement over DVFRET and CFRET, eliminating out- sensitive over FCS, in that it cross-correlates two or more of-signal issue and light-induced bleaching and damage fluorescent channels to study molecular interactions (Obeng drawbacks, but at the same time produced spectral cross-talk et al. 2016). Fluorescence recovery after photo-bleaching bleed-through noise signals. Then, lifetime imaging FRET (FRAP) is an alternative to FCS that images molecular (LFRET) microscopy was developed that minimized the kinetics such as diffusion and transport. In FRAP, a partic- spectral noise as a result of overlap between the donor and ular region is photobleached and the recovery of fluores- acceptor fluorescent molecules. LFRET provided high spa- cence over time is monitored due to movement of tial and temporal images of cellular events in a dynamic surrounding non-bleached fluorophores into the bleached manner. area generating images. Other techniques that are being Exploring interconnected intra- and inter-biological events exploited these days are fluorescence lifetime imaging within the natural environment of a cell via spectroscopic (FLIM) that maps the molecular environment of fluorescent- advancements has become indispensable in making the tagged biological molecules by exploiting the fluorescence future of FRET very bright. There are innumerable spec- lifetime of the fluorophores; FLIM-FRET for studying troscopic imaging techniques that are available nowadays, molecular interactions and conformational changes in a Role of green fluorescent proteins in development of FRET-based sensors 775

Excitaon of CFP results in transfer of energy to YFP which then emits light and shows fluorescence Ro Energy (E)

Excitaon of CFP does not results in fluorescence of YFP Distance (R)

Figure 11. Dependence of FRET efficiency on the Fo¨rster distance.

protein; fluorescence loss in photobleaching (FLIP) repeat- molecule (Ishikawa-Ankerhold et al. 2012; Obeng et al. edly bleaches a particular area and measures the significant 2016). Bimolecular fluorescence complementation (BiFC) is decline in fluorescence intensity outside the bleached area another method used to study protein–protein interaction and which is attributed to the diffusion of bleached, non-fluo- compartmentalization of protein complexes within cells. rescent molecules within the cell; fluorescence localization GFP variants, dihydrofolate reductase, b-lactamase and after photobleaching (FLAP) uses one photobleached fluo- luciferase are used as reporter proteins in BiFC that produces rescent molecule and the other a reference molecule for fluorescence only on interaction of two proteins (Wong and tagging a protein that allows tracking the fate of the labelled O’Bryan 2011).

(a) Binding of a metabolite

D A DA

D

(b) Protease-acvated FRET-based sensor D A A

A (c) Protein-protein interacon

D D: Donor fluorophore D A A: Acceptor fluorophore

Figure 12. Three categories of FRET-based biosensors. 776 Neha Soleja et al. This is the era of ‘super-resolved fluorescence micro- Structural PALM imaging is used to detect the molecular scopy’. For illumination-based techniques, a Nobel Prize in distribution of PA-FPs with the cell which requires fixing of chemistry 2014 was jointly awarded to Eric Betzig, Stefan cells. Two-photon irradiation of optical sections of sample Hell and William E. Moerner. This breakthrough marked the along with super-resolution imaging provided by PALM is advancement that surpassed the Abbe’s limit of optical employed to deeper visualization of cells and tissues. Two- microscopy and opened the door for scientists to the nano- colour PALM allows labelling of different proteins within the world. Two alike but distinct techniques were highlighted. same cell using different pairs of PA-FPs that enables visu- One was STimulated Emission Depletion (STED) micro- alization and creation of highly resolved images. PALM/ scopy developed by Stefan Hell in 2000 that scans cell STORM techniques are increasingly being used to improve images by utilizing two concentric laser beams. The other the axial resolution in diffraction-limited imaging. Interfer- technique that fetched Eric Betzig and William Moerner, a ometric photoactivated localization microscopy (iPALM) shared Nobel Prize was single-molecule microscopy. The uses self-interference of photons from a photon emitter (i.e. scientists were successful in creating dense superimposed PA-FP). The three-way output beams from the splitter are images at nanoscale by turning the fluorescence of individual then used to provide 10-fold improvement in determining the molecules of the same area on and off multiple times axial (z) position of the molecules. PALM has recently made allowing a few interspersed molecules fluoresce each time possible imaging of live cells for tracking of single PA-FP- (Mo¨ckl et al. 2014). Probe-based super-resolution techniques tagged molecules. Single particle tracking PALM (sptPALM) such as photoactivated localization microscopy (PALM), involves photoactivating numerous molecules and tracking fluorescence photoactivation localization microscopy them until they are photobleached (Lippincott-Schwartz and (FPALM) and stochastic optical reconstruction microscopy Patterson 2009). (STORM) became increasingly popular techniques that make Attempts were then made to replace GFP variants by FPs use of photoswitchable fluorescent molecules to photoacti- based on LOV (light, oxygen or voltage sensing) domains. vate individual molecules to a bright ‘on’ state, which are These domains belong to the family of Per-ARNT-Sim then imaged and photobleached. Thus, overcoming light’s (PAS) proteins and offer benefits such as smaller size, diffraction barrier that helps resolve dense population of effectiveness under low-oxygen conditions and shows pH closely spaced molecules that would otherwise be spatially and thermal stability (Buckley et al. 2015). With the revo- indistinguishable. Repeated cycles of photoactivation, lution in imaging approaches, scientists are becoming suc- imaging and bleaching create single-molecule positions that cessful in surpassing the limitations of optical microscopes are merged to generate a super-resolution image. The change and enhancing the resolution of cellular events within the in the fluorescence of photoactivated PA-FPs before and after living cells in real time. photoactivation is defined as the contrast ratio. Imaging FRET-based biosensors can be categorized into three purposes require brighter PA-FPs with high-contrast ratios, groups (Palmer et al. 2011) (figure 12): which in the absence of controlled activation allows spon- 1. Binding of a metabolite to a periplasmic-binding protein taneous photoconversion, thereby increasing the background (PBP) elucidates the conformational change that fluorescence to produce a super-resolved image. PA-FPs increases the FRET efficiency between the two GFP photobleach readily; thus, limiting the time period for which variants. For example: cameleon Ca2?-sensors as well the signals can be observed. Single molecule super-resolution as sensors for sugars, glutamate, leucine, Zn2?, cAMP, imaging requires optimal balance between the rate of pho- cGMP, NO and membrane potential. tobleaching and photoactivation. Photoactivating molecules 2. Protease-activated FRET-based sensors: the protein before bleaching of the already localized molecules results in when activated by enzyme cleaves a certain sequence overlapping signals from PA-FPs making molecules spatially that leads to a reduction of FRET between the donor less indistinguishable and would not be able to surpass the and acceptor as the distance of FPs increases from each light’s diffraction limit. PALM and FPALM used photoacti- other like caspases and matrix metalloproteases. vatable or photoconvertible FPs (tandem dimer and 3. Protein–protein interaction-based sensors: a receptor is photoactivatable GFP (PA-GFP)) as the switchable probes, tagged with a donor fluorophore and hormone with an whereas STORM was developed using synthetic dyes, Cy3 acceptor fluorophore. When the hormone binds to the and Cy5 as labelling probes. Molecules spaced within their receptor, the two fluorophores come close together resulting diffraction-limited spots are photoactivated to record the in FRET. distribution of photons in that particular spot in order to produce super-resolved image. The recorded distributed The basic principle behind construction of genetically enco- pattern of photons is then fit statistically to determine the ded FRET-based biosensors is the presence of ligand-binding precise location of the molecules. Single molecule detection proteins which act as a ligand sensing domain. Then the two is carried out by imaging using total internal reflection fluo- suitable GFP variants are attached on N- and C-termini of the rescence (TIRF) microscopy and a highly sensitive camera. ligand sensing domain. When a ligand binds to this domain, the Role of green fluorescent proteins in development of FRET-based sensors 777 two variants come in close proximity, resulting in the alteration of domain that gave rise to another calcium indicator, GCaMP3. FRET efficiency (Mohsin et al. 2015). Further improvements in GCaMP led to the development of In 1998, Tsien et al. reported FRET between two FPs linked improved, brighter, more photostable calcium indicators such by calmodulin. The donor–acceptor distance provides infor- as GCaMP5, GCaMP6, fastest GCaMP6f, GCaMP7 and mation about the location of the CaMs (calmodulin) within the GCaMP8 (Badura et al. 2014). Researchers engineered a ion channel. This sensor can be used to monitor the calcium genetically encoded FRETsensor for phosphate (Pi) by fusing a level in muscles (Rizzo et al. 2004). It was the first FRET-based phosphate-binding protein to eCFP and Venus, respectively, at sensor to be developed. After development of FRET-based the N- and C-termini. Purified fluorescent indicator protein for sensors for calcium ions, the trend of creating various other inorganic phosphate (FLIPPi), in which the fluorophores were sensors continued and gained much popularity among attached to the same PiBP lobe, showed Pi-dependent increases researchers owing to its usefulness in real-time spatio-temporal in FRET efficiency (Gu et al. 2006). A FRET-based lysine visualization in living cells. A FRET-based genetically encoded sensor (FLIPK) was created by sandwiching lysine binding sensor has been developed to measure zinc, where CFP and periplasmic protein (LAO) between two GFP variants, CFP and YFP were used as the donor and acceptor pairs and fused at the YFP. This genetically encoded nanosensor had a Kd of 97 lM N- and C-termini of SmtA from Synechococcus sp. (Nguyen and can be used to monitor the level of lysine in vivo during the and Daugherty 2005). FLIP-Leu was another genetically fermentation process (Ameen et al. 2016). A biosensor for encoded sensor that was constructed to detect the intracellular determining the role of neutrophil elastase (NE) in inflamma- level of leucine in plants and animals by flanking CFP and YFP tory tissues was created by fusing the NE-sensing domain with to LivK, a leucine binding protein from E. coli (Mohsin et al. two GFP variants. FRET occurs when the two variants 2013). Another addition to the FLIP series was FLIPM, a sensor approached each other in the uncleaved form whereas a for monitoring the level of methionine in bacterial and yeast decrease in the FRET ratio is observed upon cleavage by NE cells by sandwiching MetN between CFP and YFP (Mohsin (Schulenburg et al. 2016). Spatiotemporal monitoring of and Ahmad 2014). A series of genetically encoded FRET-based metabolites was emphasized in developing FRET-based nanosensors, eZinCh-2, ER-eZinCh-2, eCALWY-4 and ER- biosensors as it gives a better and a detailed account of the cell eCALWY4, was developed for zinc ions that uses Cerulean and signalling pathways in order to study diseased conditions and Citrine as fluorescent pairs to monitor cytosolic Zn2? levels and drug targets (Ivanova et al. 2016). Protein kinases play a release of Zn2? ions from ER via ZIP7, a Zn2?-specific ion prominent role in biological processes involving cell signalling channel. Another addition to the series was redCALWY that pathways. Genetically encoded biosensors specific for these uses red-shifted mOrange and mCherry as FRET pairs in place enzymes were created to monitor the flux of these enzymes at of Cerulean and Citrine. These sensors were designed to the sub-cellular level (Gonza´lez-Vera and Morris 2015). Efforts determine the concentration of zinc ions in and ER of are being made to develop better donor–acceptor pairs to breast cancer cell lines, MCF-7 and TamR. Treatment of these overcome certain limitations. mClover3 and mRuby3 is one cell lines with external Zn2? and an ionophore, pyrithione such pair in which mClover3 improves photostability by 60% resulted in ZIP7-mediated release of Zn2? from the ER to the and mRuby3 by 200%. Also, mRuby3 is known to be 35% cytosol whereas no change in intracellular Zn2? levels was brighter than mRuby2 (Bajar et al. 2016). Recently, flow observed when treated with EGF/ionomycin (Hessels et al. cytometry has been employed for multiplexed FRET mea- 2016). CLY9-2His and Cys2His2 are the other two FRET-based surements in cells expressing different sensors. They created zinc sensors created (Dittmer et al. 2009). For FRET-based polyclonal transfectant lines, each expressing a different magnesium sensors, the Mg2? binding domain of human cen- intramolecular FRET sensor and using flow cytometry they trin 3 (HsCen3) was fused with Cerulean and Citrine fluorescent could identify the protein kinase C (PKC)-activating compound domains at the N- and C-termini to yield MagFRET-1, which teleocidin A-1 containing well (Doucette et al. 2016)(table3). 2? combines a physiologically relevant Mg affinity (Kd=148 To lessen spectral bleed-through, a dual FRET-based caspase-3 mM) with a 50% increase in emission ratio upon Mg2? binding biosensor was developed for comparing the delay in the onset of due to a change in FRETefficiency between cerulean and citrine caspase-3 activity between the cytoplasm and nucleus during (Lindenburg et al. 2006). Recently, a family of GFP–calmod- apoptosis. Asp-Glu-Val-Asp (DEVD), a caspase substrate was ulin (GCaMP) was designed as indicators for fused between the two FPs of the respective FRET pairs, determining Ca2? levels involved in neuronal networks linked mCitrine-DEVD-mTFP1 and mAmetrine-DEVD-tdTomato to information processing. The first in the family, GCaMP1 was resulting in 42% and 45% of FRET efficiencies and emission designed using a circularly permuted EGFP domain with ratio change from 1.35 to 0.55 and 0.67 to 0.22 during prote- linkers that connected the M13 peptide and a CaM domain to olysis of these constructs (Ai et al. 2008). A FRET-based the N- and C-termini, respectively. It was shown to bind Ca2? nanosensor, Laconic was designed to measure the concentration ions with Kd of 235 nM but suffered from limitations such as and flux of lactate in mammalian cells, Warburg effect and to poor expression at 37°C and high pH sensitivity. The M13 monitor whether a particular cell utilizes or generates lactate sequence was then replaced with the CaM binding RS20 using a monocarboxylate transporter (MCT) blocker. LldR, 778 Table 3. List of some popular biosensors utilizing GFP and its variants as reporter proteins

Sensor target Type Specificity Sensor name Sensor type Reporter proteins Sensor design References Enzyme Non- H4 lysine Histac-K12 (K12) FRET-based CFP, Venus Ito et al.(2011) Activity Kinase biosensors Histone lysine K9 reporter FRET-based CFP, YFP Lin et al. biosensors (2004) O-GlcNAc transferase O-GlcNAc sensor FRET-based eCFP, Venus Carrillo et al. biosensors (2006)

Various proteases donor FP- FRET-based eCFP, eBFP Xu et al. substrate- biosensors (1998) acceptor FP Kinase Extracellular signal- Miu2 FRET-based CFP, YFP Fujioka et al. regulated kinase ERK) biosensors (2006) Insulin Receptor Phocus-2pp FRET-based eCFP, eYFP Sato et al. biosensors (2002) Protein kinase A AKAR1-34 FRET-based Various Baird et al. biosensors (1999)

Protein kinase B/Akt Aktus FRET-based CFP, YFP Sasaki et al. al. et Soleja Neha biosensors (2003)

Protein kinase D DKAR FRET-based mCFP, mYFP Kunkel et al. biosensors (2007)

Abl Picchu-Z/EGFR-Z FRET-based CFP, YFP Kurokawa biosensors et al.(2001)

Src Srcus FRET-based CFP, YFP Wang et al. biosensors (2005)

CyclinB-Cdk1 CyclinB-Cdk1 FRET-based CFPmCerulean), Gavet and sensor biosensors YFPYPet) Pines (2010) Epidermal growth factor EGFR-ECFP/PTV- FRET-based eCFP, eYFP Offterdinger receptor (EGFR) EYFP biosensors (citrine) et al.(2004) Table 3. continued

Sensor target Type Specificity Sensor name Sensor type Reporter proteins Sensor design References Ions pH GFpH/ Single FP-based Awaji et al.(2001) YFpH intrinsic biosensors

PO42- FLIPPi FRET-based eCFP, Venus Gu et al.(2006 sensors FRET-based of ) development in proteins fluorescent green of Role biosensors

Zn2? Cys2His2 FRET-based CFP, YFP Dittmer et al. biosensors (2009)

Ca2? Case12/16 Single FP-based cpFP Baird et al.(1999) extrinsic biosensors Cameleons FRET-based Miyawaki et al. biosensors (1997)

Cl- Cl-sensor FRET-based mutated eYFP, Markova et al. biosensors CFP (2007) Cu? Ace1/Mac FRET-based CFP,YFP Wegner et al. 1-FRET biosensors (2011)

Hg2? IFP/BV Single FP-based Gu et al.(2011) sensor intrinsic biosensors Cd2? Cd-FRET-2 FRET-based Vinkenborg et al. biosensors (2011)

Sugars Sucrose FLIPsuc FRET-based eCFP, eYFP Lager et al.(2006) sensors biosensors Glucose AcGFP1- FRET-based AcGFP1, Veetil et al.(2010) GBPcys- biosensors mCherry mCherry Maltose FLIPmal FRET-based eCFP, eYFP Kaper et al.(2008)

sensors biosensors (venus) 779 780 Table 3 (continued) Sensor target Type Specificity Sensor name Sensor type Reporter proteins Sensor design References

Amino acids Glutamate FLIPE FRET-based eCFP, Venus Deuschle et al. sensors biosensors (2005) Histidine FLIP-HisJ/ FRET-based eCFP,Venus Okada et al.(2009) cpHisJ194 biosensors

Leucine FLIP-Leu FRET-based CFP, YFP Mohsin et al. biosensors (2013) Methionine FLIPM FRET-based CFP, YFP Mohsin and biosensors Ahmad (2014) Secondary Diacylglycerol Daglas FRET-based CFP, YFP Nishioka et al. Metabolites biosensors (2008)

PtdIns(3,4,5)P3 Pippi- FRET-based CFP,YFP Yoshizaki et al. PI(3,4,5)P3 biosensors (2006) eaSlj tal. et Soleja Neha NO FRET-MT FRET-based Pearce et al.(2000) biosensors Energy NADH Peredox Single FP-based cpT-Sapphire Hung et al.(2011) extrinsic biosensors ATP ATeams FRET-based CFP (mseCFP), Imamura et al. biosensors YFP (cp173- (2006) mVenus)

Protein Rho family GTPase Raichu- FRET-based CFP, YFP Mochizuki et al. Activation and activation RalA biosensors (2001) Conformation

Akt conformation ReAktion FRET-based eCFP, Citrine Ananthanarayanan biosensors et al.(2007) Estrogen receptor ligand CEY FRET-based seCFP, eYFP De et al.(2005) binding domain biosensors conformation Voltage and Voltage FlaSh Single FP-based Guerrero et al. Redox extrinsic (2002) biosensors

Redox HSP-FRET FRET-based CFP, YFP Waypa et al. biosensors (2006) Role of green fluorescent proteins in development of FRET-based sensors 781 comprising a lactate binding/regulatory domain and a DNA- various molecular and biochemical events in the living cells. binding domain, was sandwiched between two fluorescent Researchers are trying to move towards the far-red and near- pairs, mTFP and Venus that sense lactate in the range of 1 lM– infrared end of the spectrum to create FPs. The curiosity 10 mM and reveals three to fivefold higher production of lactate about understanding the pathways inside the living cell has in T98G glioma cells than in astrocytes and gives values of encouraged scientists to make new and improved variants of Warburg effect as 4.1±0.5 for T98G glioma cells and these fluorescent probes. Since the use of fluorescent dyes 0.07±0.007 for astrocytes (Martin et al. 2013). Genetically has been suspended due to their toxicity and the genetically encoded FRET-based nanosensors for visualizing the concen- encoded FPs have minimized the utilization of fluorescent tration of vitamin B12 in living cells was developed by fusing dyes and focus has shifted towards the fluorescent proteins vitamin B12 binding protein, BtuF with cyan CFP and YFP at N- technology which is flourishing with immense success and and C-termini, respectively. SenVitAL (Sensor for Vitamin may become an increasingly important imaging technique in Anemia Linked), the constructed nanosensor measures vitamin the coming future. Since the last decade, the use of FPs B12 levels in a concentration-dependent manner with an affinity tremendously increased in the engineering of sensors, a constant, Kd of *157 lM and monitors the flux of vitamin B12 number of genetically encoded FRET-based tools have been levels in yeast and mammalian cells (Ahmad et al. 2018). created rapidly and will continue to be one of the hottest growing research areas in the future. The success of these fluorescent probes suggests that almost any of the biological, chemical or molecular events can be monitored using the 9.2 Limitations of FPs and possible FRET challenges appropriate FRET-based biosensor. 1. Fusion of FPs as tag represents an addition to the pro- tein molecule rather than a small modification to the Acknowledgements protein, thereby increasing the overall size that may offer steric hindrances for protein folding and their The first author (NS) is thankful to University Grants functions and expressions. Commission for a Junior Research Fellowship. Financial 2. Improper choice of FPs may result in ‘unplanned’ assistance in the form of a start-up research grant (Grant No. homo-fretting (occurs between identical fluorophores), YSS/2014/000393/LS) from SERB, Department of Science spectral crosstalk (or spectral bleed-through) and back- and Technology, Government of India for conducting this ground autofluorescence problems. research work is gratefully acknowledged. 3. Rate of chromophore maturation of different FPs (GFP shows slow maturation) may provide constraints on the utility of these molecules in certain cases. For posttransla- tional cyclization of its fluorophore, GFP requires molecular References oxygen which limits its usage under tumorous conditions. 4. 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Corresponding editor: AMITABHA CHATTOPADHYAY