Received 6th February 2013,Accepted 12th April 2013
DOI: 10.1039/c3pp50040c
www.rsc.org/pps
Advanced in vivo applications of blue lightphotoreceptors as alternative fluorescent proteins
Thomas Drepper,*a Thomas Gensch*b and Martina Pohlc
The ultimate ambition in cell biology, microbiology and biomedicine is to unravel complex physiological
and pathophysiological processes within living organisms. To conquer this challenge, fluorescent proteins
(FPs) are used as versatile in vivo reporters and biosensors to study gene regulation as well as the syn-
thesis, localization and function of proteins in living cells. The most widely used FPs are the green fluo-
rescent protein (GFP) and its derivatives and relatives. Their use as in vivo reporter proteins, however, is
sometimes restricted by different environmental and cellular factors. Consequently, a whole range of
alternative, cofactor-dependent reporter proteins have been developed recently. In this perspective, we
summarize the advantages and limitations of the novel class of cyan-green fluorescent flavoproteins in
comparison to members of the GFP family and discuss some correlated consequences for the use of FPs
as in vivo reporters.
Genetically encoded fluorescent proteins, like GFP from thejellyfish Aequorea victoria, have undergone a long history ofoptimization to become one of the most variable and popularin vivo reporters in cell biology, microbiology and biomedicine.The relatively simple and self-assembling FPs can be expressedin a broad range of different organisms ranging from bacteriato mammals. They can easily be detected by non-invasivefluorescence techniques and function without additional pros-thetic groups. GFP-like proteins can therefore be used tomonitor the expression, folding, localization, movement, and
Thomas Drepper
Thomas Drepper studied biologyand since 2004 he has been headof the Bacterial Photobiotechnol-ogy group at the Institute forMolecular Enzyme Technology,Heinrich-Heine-University Düs-seldorf, Germany. His scientificinterests lie in the analysis andapplication of light-dependentprocesses. In particular, hisgroup developed methods to usephotosynthetic bacteria for bio-technological applications. Inaddition his lab established
novel fluorescent reporter proteins and biosensors based on bac-terial blue-light photoreceptors.
Thomas Gensch
Thomas Gensch studied physicsand works since his diploma(1992), in different photo-physical and photobiologicalresearch areas. Since 2000, hehas been working in the For-schungszentrum Jülich (Instituteof Complex Systems 4 (CellularBiophysics)) being responsiblefor various modern fluorescencemicroscopy methods and theirapplication in cell biology. Hisresearch includes protein confor-mation studies, photophysics of
fluorescent proteins and dyes, fluorescent sensor development forenvironmental parameters towards spatially and time resolvedmultiparameter measurements in living cells and single moleculefluorescence applications.
aInstitute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf,
interaction of proteins in live cells and tissues (e.g. ref. 1).Furthermore, color variants of GFP can be used to generategenetically encoded FRET-based biosensors for the non-inva-sive detection of intracellular factors including Ca2+, Zn2+, Cl−,pH, ATP, cAMP, cGMP, IP3, and different sugars as was forexample comprehensively reviewed by Newman et al.2 Becauseof the versatile applicability of GFP for in vivo imaging andquantitative analyses, in 2008 the Nobel Prize in chemistry wasawarded to Shimomura, Chalfie and Tsien.3–6
Potential limitations of GFP-based in vivoapplications
Due to the absence of signal amplification, the applicability offluorescent proteins as in vivo reporters is generally limited bytheir brightness that, in turn, is directly coupled to the sensi-tivity of the used instrument and detection method. Thein vivo fluorescence brightness of all FPs is basically deter-mined by three fundamental aspects: (1) the intrinsic photo-physical properties of the respective FP, including itsextinction coefficient and fluorescence quantum yield, (2) theinteraction of FPs with a variety of individual biological pro-cesses that take place in the respective organism as well as (3)the presence and local alteration of environmental and cellularfactors that might affect the FP characteristics. Thus, in livingorganisms the FP-mediated fluorescence is the ‘final product’of a complex multi-step process that is affected on variouslevels (Fig. 1). In the following, some of these aspects concern-ing maturation and environmental sensitivity that limit theuse of FPs as in vivo reporters are briefly discussed.
Basically, GFP-like FPs have to pass two different matu-ration steps, i.e. the protein folding process and the autocataly-tic chromophore formation, before the fluorescence signal canbe detected.7 The process of FP maturation starts with theinitial protein folding resulting in the formation of the charac-teristic β-barrel fold,8 before the chromophore is subsequentlyformed in an autocatalytic multistep reaction.7,9–11 However,in bacteria, for example, the folding process of GFP and manyof the engineered FP variants and relatives is sometimes
inefficient (e.g. ref. 12–19). In addition, the folding yield mightbe further reduced when FPs are expressed at higher tempera-tures (e.g. 37 °C)20,21 or fused to hydrophobic or poorly foldingproteins.14,22,23 As a consequence, FPs can either be degradedor might accumulate in non-fluorescent forms.24–26
In addition to the FP inherent folding properties, thefolding efficiency can further depend on different factors givenby the respective biological system. For instance, the foldingcapacity of pro- and eukaryotic cells for FPs differ signifi-cantly.27,28 As shown by comparative expression analyses thefolding efficiencies of both, GFP derivatives and recombinantGFP fusions, are remarkably lower in E. coli than in yeast andMartina Pohl
Martina Pohl studied chemistryand since 2009 she has beenhead of the Biocatalysis & Bio-sensors group at the IBG-1: Bio-technology at ForschungszentrumJülich, Germany. Her researchfocus is on enzymes for the syn-thesis of chiral fine chemicalsand the development of geneti-cally encoded biosensors forwhole cell applications.
Fig. 1 Potential limitations of GFP-like FP synthesis and function. The modelsummarizes aspects that can limit the use of GFP-like FPs as in vivo reporter pro-teins. The (1) expression efficiency of a reporter gene is influenced by thestrength of the controlling promoter (Ptarget), as well as by limitations that mightoccur on the level of transcription and translation. (2) Maturation of GFP-like FPsinvolves protein folding and chromophore formation. The latter strictly dependson molecular oxygen. (3) The spectral properties of the chromophore of matureFPs including its fluorescence brightness can significantly be affected by variouscellular and environmental factors. The most important aspects discussed indetail are marked in red.
mammalian cells. The authors proposed that the foldingmechanism during translation as well as the properties of cel-lular chaperones considerably diverge in bacteria compared toeukaryotes. Therefore, the overall in vivo process of (chaper-one-assisted) FP folding, which is directly coupled to the quan-titative development of its fluorescence, can be affected by thefolding properties of the reporter protein and eventually of itsfusion partner.
Furthermore, GFP and its derivatives do not fluoresceimmediately after protein folding. The subsequent autocataly-tic maturation process of the chromophore includes cycliza-tion and oxidation of an internal amino acid triplet (e.g. in thecase of GFP Ser–Tyr–Gly at positions 65–6729). Basically,chromophore formation of all FPs involves either one (for allGFP related proteins) or two (for DsRed and its homologuesand variants) final oxidation steps.10 Early in vivo and in vitroanalyses demonstrated that the final oxidation steps ofchromophore formation resulted in a relatively slow rate offluorescence development: When GFP is expressed in E. coliunder anaerobic conditions, protein accumulation but notGFP-mediated fluorescence is detectable. After exposure toatmospheric oxygen, fluorescence of GFP was observed after acertain delay.26,30 Similar observations have been made inin vitro time course experiments, where the chromophores ofmature FPs were reduced by strong reducing agents and, sub-sequently, the kinetics of the reoxidation process were moni-tored by the reacquisition of FP fluorescence.24,31 In the caseof DsRed and its derivatives, acquisition of its red fluorescenceproceeds in a two-step process involving the formation of apremature green fluorescent species after a first oxidation stepand a modification of the fluorophore precursor via a secondoxidation reaction resulting in the fully matured red FP. Thus,the overall maturation process of orange and red FPs is inher-ently slower compared to the GFP-like proteins (e.g. ref. 19,32–36), although faster maturing variants like mCherry andE2-Crimson were also described.37
In addition to the processes that influence the synthesisand maturation of GFP-related FPs, their utilization as in vivoreporter proteins can further be limited by different environ-mental factors affecting their signal intensity. Well known arethe pH- and halide sensitivities of some GFP-like FPs. pH-sen-sitivity of the wild type GFP, for example, is caused by the highpKa of the p-hydroxybenzylidene-5-imidazolinone (p-HBI)chromophore formed by Ser65, Tyr66 and Gly67, which isstrongly influenced by the hydrogen-bonding network of thesurrounding protein (see ref. 38 and references therein). Inthis context, especially some of the yellow GFP variants such asEYFP exhibit very strong pH (pKa of EYFP ≥7) and halidedependencies (see ref. 2 and 39 and references therein). pH-sensitivity of these FP derivatives particularly affects theirapplicability as in vivo reporters, if their fluorescence is reversi-bly quenched by acidic pH that might occur in compartmentsor organelles of living cells. Besides, different variants exhibit-ing increased pH tolerance have also been constructed. Amongthese, Citrine (EYFP-V68L/Q69M; pKa = 5.7)40 and Venus(EYFP-M153T/V163A/S175G; pKa = 5.8)41 are now frequently
used. Concerning the halide sensitivity observed with YFP-vari-ants Wachter et al. identified a halide binding site at His148,which is located next to the chromophore.42,43 The low halidesensitivity of Citrine was attributed to the Q69M substitution,which plugs an anion binding pocket next to the chromo-phore.40
Apart from these well known influences on the fluorescenceproperties of GFP-related fluorescent proteins, further effectsof other environmental factors (e.g. various anions such asfluoride, bromide, iodide, nitrate, perchlorate, formate andthiocyanide as well as ATP and hydroxyradicals) were rarelydescribed for certain GFP variants.42,44,45 These studiessuggest that the factors which might affect the fluorescenceproperties of in vivo reporters are much broader than currentlyknown. This is also indicated by our recent investigationswhich further demonstrated the strong influence of other cel-lular components including nicotinamide cofactors on thefluorescence properties of EYFP and Citrine (M. Pohl, unpub-lished results).
LOV-based fluorescent reporter proteins
Fluorescent proteins of the GFP family are widely used toquantitatively monitor protein dynamics in live cells. However,as discussed above, the relatively slow maturation of these FPvariants (∼0.5 h to several hours) as well as their environ-mental sensitivities can lead to a discrepancy between thequantifiable fluorescence signal and the amount of accumu-lated reporter protein at a given time-point. As a consequenceof these limitations, a whole range of alternative, cofactor-dependent reporter proteins have been developed recently.Besides the group of NADPH-binding blue-fluorescentproteins46–49 and bilin-dependent red-fluorescingreporters,50–56 a family of cyan-green fluorescent reporter pro-teins that bind flavin nucleotides as chromophores has gainedparticular attention.
All members of the new class of flavin-dependent FPs are,in terms of light perception, photochemically inactive deriva-tives of LOV (light oxygen voltage) photoreceptor proteins.57–61
The novel FPs, designated as FbFPs (FMN-binding fluorescentproteins;62 trademark name: evoglow) and iLOV,63 were engin-eered either from Bacillus subtilis YtvA (BsFbFP and the trun-cated derivative EcFbFP adjusted to E. coli codon bias),64
Pseudomonas putida SB2 (PpFbFP)65 or Arabidopsis thalianaphototropin 2 LOV2 domain (iLOV),63 respectively. These FPsare notably small (∼11–16 kDa), form either monomers ordimers and harbour the conserved global LOV/PAS fold (i.e.the core LOV domain, Fig. 2A) as a functional domain. Thecore LOV domain consists of a five-stranded antiparallelβ-sheet and four short helices58,66–70 and predominantly bindsflavin mononucleotide (FMN) as a chromophore, which is ubi-quitously distributed and thus can be provided by any hostorganism without additional coexpression of the corres-ponding cofactor biosynthesis genes. The FMN chromophoreis cooperatively coordinated by different amino acid residues
located inside the β-sheet and two of the four helices. TheLOV-based FPs show a cyan-green fluorescence (λmax = 495 nm)upon excitation with blue light (λmax = 450 nm) which is insen-sitive towards acidic pH (Fig. 2B). Extensive mutational andstructural analyses demonstrated that changes of the FMNenvironment, further constraining the chromophore, led to anincreased fluorescence brightness and photostability.68
However, in contrast to many of the GFP variants, brightnessof LOV-based FPs is still comparatively low with an extinctioncoefficient of approximately 12 500 M−1 cm−1 and fluorescencequantum yields of up to 44% (EcFbFP: 0.39; PpFbFP: 0.17;PpFbFP variant F37S: 0.30; iLOV: 0.44).62,63,71,72
LOV-based fluorescent proteins as oxygen-independent in vivoreporters
Initially, LOV-based FPs have been described as the first fluo-rescent in vivo reporters that can be used in the presence andabsence of molecular oxygen.62 This is of particular impor-tance, because the O2-dependent chromophore formation ofconventional jellyfish FPs clearly limits their use for in vivoanalyses of physiological, pathological and biotechnologicalprocesses that are influenced by oxygen limitation. Since thesefirst studies, many groups have found these “anaerobic fluo-rescence reporter proteins” useful for a variety of differentapplications: FbFP was successfully utilized as an in vivo fluo-rescence reporter (1) in the photosynthetic bacteria Rhodobac-ter capsulatus and Roseobacter denitrificans, which can generateenergy via anoxygenic photosynthesis,62,73 (2) in two non-phototrophic members of the Roseobacter clade,73 (3) in non-pathogenic and pathogenic E. coli strains,74,75 (4) in the obli-gate anaerobic bacteria Porphyromonas gingivalis, Bacteroidesfragilis and Clostridium cellulolyticum,76–78 and (5) in the facul-tative anaerobic yeasts Saccharomyces cerevisiae and Candidaalbicans.79 Remarkably, the FbFP-labelled opportunisticanaerobic pathogens P. gingivalis and B. fragilis could be visu-alized inside human host cells for the very first time by in vivoimaging techniques. The results thereby gave new insights intohost–pathogen interactions during infection of humanprimary gingival epithelial cells by P. gingivalis and internaliz-ation of B. fragilis by murine macrophage cells.76,78 Veryrecently, a comparable iLOV-based strategy was brieflydescribed to analyze the localization of the translocatedintimin receptor Tir in enterohaemorraghic E. coli cells duringhost cell infection.74 In addition, another FbFP applicationwas described by Walter et al. (2012).80 There, FbFPs have beenused for reliable imaging of living mammalian cells, includinghuman tumor and mouse neuronal stem cells, under hypoxicconditions (Fig. 2C). Consequently, as many fields of biomedi-cal research, including microbial infection, stroke researchand oncology, are associated with hypoxia, LOV-based FPsprovide a novel and so far unique access to several importantbiomedical issues.
Novel biotechnological applications
Besides the utilization of FMN-binding FPs as oxygen-indepen-dent in vivo reporters, these markers were further
Fig. 2 Properties and novel applications of flavin-binding fluorescent proteins.A: Protein structure of the YtvA core LOV domain70 (PDB: 2PR5). The core LOVdomain exhibits the canonical LOV/PAS fold. FMN is shown as a ball-and-stickmodel. B: Spectral properties of FbFP and the cyan fluorescing ECFP (blue andblack, respectively). The figure shows the absorption at pH 8.0 (left) and fluo-rescence spectra at pH 8.0 and 6.0 (right; λexc = 440 nm, normalized to thespectra at pH 8.0). C: Expression of FbFP in neuronal stem cells under oxygen-sufficient and deficient conditions. The figure originates from Walter et al.(2012) and was slightly modified.80 Critical cell structures can be visualizedunder hypoxia when FbFP is used (indicated by white arrows) whereas GFP fluo-rescence was not sufficient under these conditions (data not shown). D: Conven-tional wide-field fluorescence image and super-resolution (FPALM) image ofE. coli cells expressing the blue-light photoreceptor YtvA (scale bars 1 μm). Thefigures were reproduced from Losi et al. (2013).87
advantageously used in microbial biotechnology applicationsincluding protein synthesis as well as process validation andoptimization.
In baker’s yeast S. cerevisiae iLOV was applied as a reporterfor recombinant production of G-protein coupled receptors(GPCR).81 Expression of the recombinant human β3-adrenergicand μ-opioid receptors, both fused to iLOV, allowed monitor-ing of GPCR accumulation in different S. cerevisiae strains. Asimilar approach was employed to determine the productionof human sterol isomerase in fermenting cultures of the yeastPichia pastoris.74 Furthermore, FbFP has been used to establishan E. coli-based monophasic micro-aqueous whole-cell systemfor producing chiral cyanohydrins in a hydroxynitrile lyase-catalyzed reaction.82 By monitoring an FbFP-tagged hydroxy-nitrile lyase, the integrity of the bacterial cells within themonophasic organic solvent as well as the stability of the intra-cellular enzyme could be demonstrated.
Another major advantage of the small LOV-based FP pro-teins is that they rapidly gain the fluorescence-active confor-mation in vivo because of their fast folding kinetics and thespontaneous incorporation of the flavin chromophore. In com-parison to the yellow fluorescent GFP variant EYFP, the fastdevelopment of the FbFP fluorescence signal could clearly beproven via time-resolved online detection of both FP signalsduring E. coli batch cultivation.83 Because of the rapid proteinassembly together with the already discussed tolerancetowards hypoxia the LOV-based FPs gained increasing popular-ity as reliable real-time in vivo reporters for quantitative bio-technological approaches. For example, FbFP was utilized as areporter for recombinant protein production in order toimprove T7 RNA polymerase-dependent gene expression inE. coli in an automated high-throughput microfermentationplatform.84,85 By applying this novel high-throughput method,it was possible to evaluate the efficiency of protein productiondepending on inducer concentration, induction time andphosphate limitation.
New in vivo imaging approaches
Besides the enhanced folding efficiency, the small size ofFMN-dependent FPs also rendered them useful in investi-gating the dynamics of viral infections in plants.63 Using soph-isticated in planta imaging experiments, Christie and co-workers demonstrated the improved mobility of tobaccomosaic virus (TMV) particles in tobacco leaves. For these exper-iments, iLOV and GFP were comparatively expressed as freeFPs and as fluorescent tags fused to the viral movementprotein.
Furthermore, another fluorescent flavoprotein variant, alsoa derivative of the A. thaliana phototropin2 LOV2 domain, wasshown to efficiently generate singlet oxygen upon blue-lightirradiation.72,86 However, extensive mutagenesis of the LOV2domain, including DNA shuffling and random mutagenesisapproaches, was necessary to sufficiently increase the 1O2 pro-duction for further applications. As with all members of thisFP family, the novel LOV-FP, designated as the mini SingletOxygen Generator (miniSOG), is a versatile in vivo fluorescence
reporter with a quantum yield of 0.37. MiniSOG thus was suc-cessfully used as a fusion tag in a wide range of cells, incomplex organs like mouse brain as well as in intact nema-todes. Subsequently, miniSOG-mediated 1O2 formation wasapplied for the local photogeneration of an osmiophilicdiaminobenzidine polymer inside fixed cells that can bevisualized by electron microscopy at high resolution. This duallight-dependent activity therefore makes miniSOG a uniquegenetically encoded fluorescence tag that facilitates correlatedlight and electron microscopy.
In addition to the flavin-binding FPs, the great potential ofLOV photoreceptor proteins as photoswitchable FPs for super-resolution microscopy has also been described recently.87 Incontrast to the LOV derivatives applied so far as FPs, the utiliz-ation of photochemically active LOV domains generallyallows the controlled, light-induced switching between thefluorescent and non-fluorescent states. Losi and coworkersdemonstrated the applicability of LOV photoreceptors forFluorescence PhotoActivation Localization Microscopy(FPALM) by localizing the B. subtilis blue-light photoreceptorYtvA in living E. coli cells with an average resolution of 35 nm(Fig. 2D). Since the resolution, on the length scale of 100 nm,was well below the diffraction resolution limit of lightmicroscopy, respective bacterial substructures have clearlybeen resolved.
FbFPs as an alternative FRET donor domain
The great success of GFP and its relatives in life sciences relieson the smart use of the fluorescent entities that can bebrought into every organism living under aerobic conditions.FPs do not just report on the position of a protein of interestin a cell, but they can monitor very different cell propertieswith high spatial and temporal resolution. The so-calledgenetically encoded biosensors can be realized in two differentways: Either one exploits the sensitivity of the absorption andfluorescence properties of FPs on environmental parameters(single-FP biosensors) or the non-radiative energy transfer(Förster resonance energy transfer; FRET) between two fluo-rescent proteins of different color (i.e. the respective FRET-donor and -acceptor) is affected by changes of cellular para-meters. In early studies on the construction of geneticallyencoded biosensors for pH88–90 and Ca2+,91,92 respectively,both principles have already been demonstrated to work inliving cells. Today there are far more than 150 geneticallyencoded bisosensors in use for the non-invasive detection ofvarious intracellular compounds and other cellular propertiesincluding the redox state, cell apoptosis or protein phosphoryl-ation.2 Remarkably, about 70% of the so far described geneti-cally encoded biosensors are FRET-based. In most cases, thedonor and acceptor FPs are connected by a linker harboring ametabolite sensor domain. Upon specific interaction with thetarget molecule, the conformation of the sensor domainchanges thereby causing a distance and/or orientation changeof the donor and acceptor FP. In some FRET-based genetically
encoded biosensors, however, the specific sensitivity of one ofthe FPs itself towards different environmental factors was usedas an alternative sensing element, as mentioned above. Promi-nent examples are the clomeleons, where the absorption spec-trum of the FRET-acceptor domain (YFP) is shifted to shorterwavelengths upon Cl− association whereas the spectral pro-perties of the FRET-donor domain (CFP) are almostunaffected. The Cl−-sensitivity of YFP in turn results in adecrease of FRET efficiency thereby allowing a ratiometricreadout of the chloride concentration in living cells.93,94 Inaddition, the fluorescence decay of the donor FP is similarlyaffected (prolonged) due to the reduced FRET (see also below).
The most frequently used FP pair for FRET studies (ingenetically encoded biosensors but also for the investigationof protein–protein interactions) is CFP/YFP in particular thevariants distributed by Clontech (ECFP and EYFP). In the late1990s and early 2000s this FRET-pair was the best and mosteasily available pair and little attention was paid to the verycomplex photophysical behaviour of both ECFP and EYFP. Inparticular, although ECFP is used as the donor in the majorityof all cell biological FRET experiments, it has severe disadvan-tageous properties (as already briefly discussed above). A pHtransition of ECFP (pKa 6.4) was already described in the pub-lication that added this variant to the FP color palette, where itwas used to measure the pH inside the Golgi apparatus.90
However, the influence of this pH dependence, resulting in afluorescence decrease together with moderate spectral changesand a significant shortening of fluorescence lifetime (G. Casiniand T. Gensch, unpublished results), is completely neglectedin most FRET studies, although already small variations ofintracellular pH will influence the donor signal (Fig. 2B). AlsoEYFP shows a pH transition of its absorption and fluorescence,forming a non-fluorescent neutral chromophore at lower pHwith absorption below 400 nm.
A second limitation of FRET-based biosensors is the slowand potentially incomplete maturation of GFP-like FPs. Andalthough many GFP-like FP variants with optimized matu-ration properties are already described, the chromophorematuration may further depend on the cell type and environ-mental conditions (e.g. temperature, molecular oxygen concen-tration, intracellular pH, etc.), and the individual sensitivitiescan differ for the two FPs as discussed above. Again, this willlead to erroneous interpretations of quantitative FRET experi-ments and only a maturation efficiency of >95% allows unam-biguously to determine FRET changes of 10% and below.
Photoconversions (reversible and irreversible photoactiva-tions and photoswitching) have been found for GFP-like aswell as DsRed-like FPs.96–98 First seen as a drawback and curi-osity, nowadays the multitude of photoconversions are thebasis for elegant timer and migration experiments in cellbiology as well as one branch of super-resolution microscopy.Photoconversions that lead to transient or permanent colorlessforms (dark states), color-changed forms or forms withdifferent photophysical properties are likely to occur for mostFPs under the illumination conditions in modern fluorescencemicroscopes. The photoconversions of ECFP and EYFP,
however, have an unwanted influence on FRET measurements,where ECFP is transformed into a form with similar spectrabut altered fluorescence properties,99,100 while EYFP is trans-formed partially into a CFP-like species.101,102 As a conse-quence, the photoconversion phenomena of CFP and YFP canfalsify the interpretation and quantification of FRETexperiments.99,101
Due to the similar spectral properties of FbFPs and ECFP(see Fig. 2B), FbFP can principally be used as an alternativeFRET donor domain. Although the extinction coefficient islower in comparison to ECFP (FbFP: ∼12 500 M−1 cm−1; ECFP:28 000 M−1 cm−1),62,103 FbFPs do not have some of the abovedescribed disadvantageous properties of ECFP, making themgood candidates to replace ECFP in applications where pro-blems and artefacts have been detected or are anticipated. Forexample, in contrast to ECFP FbFPs are pH tolerant for pH < 7(see Fig. 2B) and no photoconversion effects are reported. Inaddition, probably no maturation issues have to be consideredfor LOV-based FPs since binding of FMN occurs very fast witha dissociation constant of 170 pM72,83 and typical intracellularFMN concentrations of ≥5 nM.
The FRET efficiencies of FbFPs to YFP are slightly highercompared to those of ECFP due to the red-shifted fluorescencespectrum and the FbFPs’ larger fluorescence quantum yield(FbFP: 0.39; ECFP: 0.36) (Fig. 2B).62,103 These unique pro-perties make FbFP an attractive alternative donor domain tobe used in genetically encoded FRET-based biosensors.
Fig. 3 FbFP as a FRET donor domain. A: Fluorescence properties of the EcFbFP-based biosensor FluBO (red) in comparison to EcFbFP (green) at an excitationwavelength specific for EcFbFP of λexc = 380 nm; a.u.: arbitrary units. B: In vivotwo-photon excitation FLIM analysis (λexc = 760 nm) of EcFbFP and FluBO. Thefigure shows single E. coli cells expressing either FbFP or FluBO. Efficient FRETreduces the in vivo fluorescence lifetime of plain EcFbFP (τave = 2.73 ns) in com-parison to EcFbFP fused to the FRET acceptor domain YFP (τave = 1.74 ns).Figures originate from Potzkei et al. (2012) and were subsequently modified.95
Recently, a FbFP-based genetically encoded biosensor,called FluBO, that allows to monitor intracellular levels ofmolecular oxygen ratiometrically has been developed andcharacterized.95 FluBO consists of FbFP as FRET-donor andEYFP as FRET-acceptor connected by a short linker. Anefficient FRET takes place in FluBO after excitation of thedonor FbFP and most of the emission occurs from the accep-tor EYFP (Fig. 3A). As a consequence, the FbFP fluorescencelifetime, analyzed by fluorescence lifetime imaging (FLIM),was significantly reduced (Fig. 3B). This, however, can onlytake place if the intracellular oxygen concentration allowsmaturation of the YFP chromophore. In E. coli cells, grownunder limited supply of molecular oxygen, chromophore matu-ration of YFP is partially or completely impaired, while FbFPfluorescence remains unaffected. Consequently, FRETefficiency of FluBO decreases under low molecular oxygen con-centrations and is completely abolished in the absence of O2.
Conclusions
GFP and its numerous variants are essential tools for manydifferent approaches in the fields of molecular and cellbiology. However, since chromophore formation and fluo-rescence brightness are distinctly affected by various environ-mental factors, novel LOV-based FPs have been developedwhich exhibit some unique and exciting properties. Because oftheir fast protein folding and fluorescence development LOV-based FPs will contribute to analyzing cellular behaviour andshort-time regulatory events as required for systems biology aswell as for the development of biotechnological processes. Fur-thermore, LOV proteins are promising fluorescent tags forhigh resolution imaging of cellular structures using light andelectron microscopy. Finally, because of their signal robust-ness and spectral properties, fluorescent flavoproteins arepromising FRET donor domains that can be used to designalternative genetically encoded biosensors.
Acknowledgements
This work was supported by grants from the Federal Ministryof Education and Research (OptoSys, FKZ 031A16).
Notes and references
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