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FMN-binding fluorescent protein

March 17, 2024

A FMN-binding fluorescent protein (FbFP), also known as a LOV-based fluorescent protein, is a small, oxygen-independent fluorescent protein that binds flavin mononucleotide (FMN) as a chromophore.

Typical core-domain of an FbFP (PDB: 2PR5​)

They were developed from blue-light receptors (so called LOV-domains) found in plants and various bacteria.[1] They complement the GFP-derivatives and –homologues and are particularly characterized by their independence of molecular oxygen and their small size. FbFPs absorb blue light and emit light in the cyan-green spectral range.

Development edit

LOV-domains are a sub-class of PAS domains and were first identified in plants as part of Phototropin, which plays an essential role in the plant's growth towards light.[2] They noncovalently bind Flavin mononucleotide (FMN) as coenzyme. Due to the bound FMN LOV-domains exhibit an intrinsic fluorescence, which is however very weak. Upon illumination with blue light, LOV-domains undergo a photocyle, during which a covalent bond is formed between a conserved cysteine-residue and the FMN. This causes a conformational change in the protein that is necessary for signal propagation and also leads to the loss of fluorescence. The covalent bond is energetically unfavorable and is cleaved with a protein specific time constant ranging from seconds to hours.[3][4][5] In order to make better use of the fluorescence properties of these proteins, the natural photocycle of these LOV-domains was abolished by exchanging the conserved cysteine against an alanine on a genetic level. Thus, upon blue light irradiation, the protein remains in the fluorescent state and also exhibits a brighter fluorescence.[1]

The first FbFPs that were generated in this fashion were subsequently further optimized using different methods of mutagenesis. Especially the brightness [6][7][8] but also the photostability [2] of the proteins were enhanced and their spectral characteristics altered.[8]

Spectral characteristics edit

 
Typical excitation and emission spectrum of FMN-binding fluorescent proteins (FbFPs)

Typically FbFPs have an excitation maximum at a wavelength of approximately 450 nm (blue light) and a second distinct excitation peak around 370 nm (UV-A light). The main emission peak is at approx. 495 nm, with a shoulder around 520 nm. One variant of Pp2FbFP (Q116V) exhibits a 10 nm blue shift in both the excitation and emission spectra.[8] Rationally designed variants of iLOV and CagFbFP exhibit 6 and 7 nm red shifts, respectively. [9]

Photophysical properties edit

The photophysical properties of the FbFPs are determined by the chromophore itself and its chemical surrounding in the protein. The extinction coefficient (ε) is around 14.200 M−1cm−1 at 450 nm for all described FbFPs, which is slightly higher than that of free FMN (ε = 12.200 M−1cm−1 [10]). The Fluorescence-Quantum yield (Φ) varies significantly between different FbFPs and ranges from 0.2 (phiLOV2.1) to 0.44 (EcFbFP and iLOV).[6][8] This represents an almost twofold increase compared to free FMN (Φ = 0.25[8]).
The difference to free FMN is even more significant in the case of the photostabaility, the proteins resistance to bleach out during prolonged and intense irradiation with blue light. Based on the bleaching-halftime (the times it takes to reduce the initial fluorescence intensity to 50% upon illumination) the genetically engineered variant phiLOV2.1[2] is approximately 40x as stable as free FMN. This stabilizing effect can be observed for almost all FbFPs, although it is usually in the range of 5x - 10x.[8]
The average fluorescence lifetime of FbFPs is in the range of 3.17 (Pp2FbFP) and 5.7 ns (e.g. EcFbFP).[8] They are thereby much longer than the ones of GFP derivatives, which are usually between 1,5 and 3 ns.[11][12] FbFPs are therefore well suited as donor domains in Förster resonance energy transfer (FRET) systems in conjunction with GFP derivatives like YFP as acceptor domains.

Advantages and disadvantages edit

The main advantage of FbFPs over GFP is their independence of molecular oxygen. Since all GFP derivatives and homologues require molecular oxygen for the maturation of their chromophore, these fluorescent proteins are of limited use under anaerobic or hypoxic conditions.[13] Since FbFPs bind FMN as chromophore, which is synthesized independently of molecular oxygen, their fluorescence signal does not differ between aerobic and anaerobic conditions.[1][14]
Another advantage is the small size of FbFPs, which is typically between 100 and 150 amino acids. This is about half the size of GFP (238 amino acids). It could for example be shown that this renders them superior tags for monitoring tobacco mosaic virus infections in tobacco leaves.[6]
Due to their extraordinary long average fluorescence lifetime of up to 5.7 ns they are also very well suited for the use as donor domains in FRET systems in conjunction with e.g. YFP (see photophysical properties). A fusion of EcFbFP and YFP was e.g. used to develop the first genetically encoded fluorescence biosensor for oxygen (FluBO)[15]

The main disadvantage compared to GFP variants is their lower brightness (the product of ε and Φ). The commonly used EGFP (ε = 55,000 M−1cm−1; Φ = 0.60 [16]) for example is approximately five times as bright as EcFbFP.
Another disadvantage of the FbFPs is the lack of color variants to tag and distinguish multiple proteins in a single cell or tissue. The largest spectral shift reported for FbFPs so far is 10 nm. Although this variant (Pp2FbFP Q116V) can be visually distinguished from the others with the human eye,[8] the spectral differences are too small for fluorescence microscopy filters.

References edit

  1. ^ a b c Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gärtner W, Jaeger KE (2007). "Reporter proteins for in vivo fluorescence without oxygen". Nat Biotechnol. 25 (4): 443–445. doi:10.1038/nbt1293. PMID 17351616. S2CID 7335755.
  2. ^ a b c Christie JM, Reymond P, Powell GK, Bernasconi P, Raibekas AA, Liscum E, Briggs WR (1998). "Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism". Science. 282 (5394): 1698–1701. Bibcode:1998Sci...282.1698C. doi:10.1126/science.282.5394.1698. PMID 9831559.
  3. ^ Circolone F, Granzin J, Jentzsch K, Drepper T, Jaeger KE, Willbold D, Krauss U, Batra-Safferling R (2012). "Structural Basis for the Slow Dark Recovery of a Full-Length LOV Protein from Pseudomonas putida". J Mol Biol. 417 (4): 362–374. doi:10.1016/j.jmb.2012.01.056. PMID 22326872.
  4. ^ Losi, A.; Gärtner, W. (2011). "Old chromophores, new photoactivation paradigms, trendy applications: flavins in LOV and BLUF photoreceptors". Photochem Photobiol. 87 (3): 491–510. doi:10.1111/j.1751-1097.2011.00913.x. PMID 21352235.
  5. ^ Crosson, S.; Moffat, K. (2001). "Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction". Proc Natl Acad Sci U S A. 98 (6): 2995–3000. Bibcode:2001PNAS...98.2995C. doi:10.1073/pnas.051520298. PMC 30595. PMID 11248020.
  6. ^ a b c Chapman S, Faulkner C, Kaiserli E, Garcia-Mata C, Savenkov EI, Roberts AG, Oparka KJ, Christie JM (2008). "The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection". Proc Natl Acad Sci U S A. 105 (50): 20038–20043. Bibcode:2008PNAS..10520038C. doi:10.1073/pnas.0807551105. PMC 2604982. PMID 19060199.
  7. ^ Mukherjee A, Weyant KB, Walker J, Schroeder CM (2012). "Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida". Journal of Biological Engineering. 6 (1): 20. doi:10.1186/1754-1611-6-20. PMC 3488000. PMID 23095243.
  8. ^ a b c d e f g h Wingen M, Potzkei J, Endres S, Casini G, Rupprecht C, Fahlke C, Krauss U, Jaeger KE, Drepper T, Gensch T (2014). "The photophysics of LOV-based fluorescent proteins - new tools for cell biology". Photochem Photobiol Sci. 13 (6): 875–883. doi:10.1039/c3pp50414j. PMID 24500379. S2CID 6068541.
  9. ^ Röllen, Katrin; Granzin, Joachim; Remeeva, Alina; Davari, Mehdi D.; Gensch, Thomas; Nazarenko, Vera V.; Kovalev, Kirill; Bogorodskiy, Andrey; Borshchevskiy, Valentin; Hemmer, Stefanie; Schwaneberg, Ulrich; Gordeliy, Valentin; Jaeger, Karl-Erich; Batra-Safferling, Renu; Gushchin, Ivan; Krauss, Ulrich (2021-04-13). "The molecular basis of spectral tuning in blue- and red-shifted flavin-binding fluorescent proteins". The Journal of Biological Chemistry. 296: 100662. doi:10.1016/j.jbc.2021.100662. ISSN 1083-351X. PMC 8131319. PMID 33862085.
  10. ^ Whitby, L.G. (1953). "A new method for preparing flavin-adenine dinucleotide". Biochem J. 54 (3): 437–442. doi:10.1042/bj0540437. PMC 1269010. PMID 13058921..
  11. ^ Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW, Royant A (2012). "Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%". Nat Commun. 3: 751. Bibcode:2012NatCo...3..751G. doi:10.1038/ncomms1738. PMC 3316892. PMID 22434194.
  12. ^ Jung G, Brockhinke A, Gensch T, Hötzer B, Schwedler S, Veettil SK (2012). Fluorescent Proteins I. From Understanding to Design Vol.11. Springer Berlin Heidelberg. ISBN 978-3-642-23371-5.
  13. ^ Drepper T, Gensch T, Pohl M (2013). "Advanced in vivo applications of blue light photoreceptors as alternative fluorescent proteins". Photochem Photobiol Sci. 12 (7): 1125–34. doi:10.1039/c3pp50040c. PMID 23660639. S2CID 39879469.
  14. ^ Walter J, Hausmann S, Drepper T, Puls M, Eggert T, Dihné M (2012). "Flavin mononucleotide-based fluorescent proteins function in mammalian cells without oxygen requirement". PLOS ONE. 7 (9): e43921. Bibcode:2012PLoSO...743921W. doi:10.1371/journal.pone.0043921. PMC 3439463. PMID 22984451.
  15. ^ Potzkei J, Kunze M, Drepper T, Gensch T, Jaeger KE, Büchs J (2012). "Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor". BMC Biol. 10: 28. doi:10.1186/1741-7007-10-28. PMC 3364895. PMID 22439625.
  16. ^ Patterson G, Day RN, Piston D (2001). "Fluorescent protein spectra". J Cell Sci. 114 (Pt 5): 837–838. doi:10.1242/jcs.114.5.837. PMID 11181166.

March 17, 2024
binding, fluorescent, protein, fbfp, also, known, based, fluorescent, protein, small, oxygen, independent, fluorescent, protein, that, binds, flavin, mononucleotide, chromophore, typical, core, domain, fbfp, 2pr5, they, were, developed, from, blue, light, rece. A FMN binding fluorescent protein FbFP also known as a LOV based fluorescent protein is a small oxygen independent fluorescent protein that binds flavin mononucleotide FMN as a chromophore Typical core domain of an FbFP PDB 2PR5 They were developed from blue light receptors so called LOV domains found in plants and various bacteria 1 They complement the GFP derivatives and homologues and are particularly characterized by their independence of molecular oxygen and their small size FbFPs absorb blue light and emit light in the cyan green spectral range Contents 1 Development 2 Spectral characteristics 3 Photophysical properties 4 Advantages and disadvantages 5 ReferencesDevelopment editLOV domains are a sub class of PAS domains and were first identified in plants as part of Phototropin which plays an essential role in the plant s growth towards light 2 They noncovalently bind Flavin mononucleotide FMN as coenzyme Due to the bound FMN LOV domains exhibit an intrinsic fluorescence which is however very weak Upon illumination with blue light LOV domains undergo a photocyle during which a covalent bond is formed between a conserved cysteine residue and the FMN This causes a conformational change in the protein that is necessary for signal propagation and also leads to the loss of fluorescence The covalent bond is energetically unfavorable and is cleaved with a protein specific time constant ranging from seconds to hours 3 4 5 In order to make better use of the fluorescence properties of these proteins the natural photocycle of these LOV domains was abolished by exchanging the conserved cysteine against an alanine on a genetic level Thus upon blue light irradiation the protein remains in the fluorescent state and also exhibits a brighter fluorescence 1 The first FbFPs that were generated in this fashion were subsequently further optimized using different methods of mutagenesis Especially the brightness 6 7 8 but also the photostability 2 of the proteins were enhanced and their spectral characteristics altered 8 Spectral characteristics edit nbsp Typical excitation and emission spectrum of FMN binding fluorescent proteins FbFPs Typically FbFPs have an excitation maximum at a wavelength of approximately 450 nm blue light and a second distinct excitation peak around 370 nm UV A light The main emission peak is at approx 495 nm with a shoulder around 520 nm One variant of Pp2FbFP Q116V exhibits a 10 nm blue shift in both the excitation and emission spectra 8 Rationally designed variants of iLOV and CagFbFP exhibit 6 and 7 nm red shifts respectively 9 Photophysical properties editThe photophysical properties of the FbFPs are determined by the chromophore itself and its chemical surrounding in the protein The extinction coefficient e is around 14 200 M 1cm 1 at 450 nm for all described FbFPs which is slightly higher than that of free FMN e 12 200 M 1cm 1 10 The Fluorescence Quantum yield F varies significantly between different FbFPs and ranges from 0 2 phiLOV2 1 to 0 44 EcFbFP and iLOV 6 8 This represents an almost twofold increase compared to free FMN F 0 25 8 The difference to free FMN is even more significant in the case of the photostabaility the proteins resistance to bleach out during prolonged and intense irradiation with blue light Based on the bleaching halftime the times it takes to reduce the initial fluorescence intensity to 50 upon illumination the genetically engineered variant phiLOV2 1 2 is approximately 40x as stable as free FMN This stabilizing effect can be observed for almost all FbFPs although it is usually in the range of 5x 10x 8 The average fluorescence lifetime of FbFPs is in the range of 3 17 Pp2FbFP and 5 7 ns e g EcFbFP 8 They are thereby much longer than the ones of GFP derivatives which are usually between 1 5 and 3 ns 11 12 FbFPs are therefore well suited as donor domains in Forster resonance energy transfer FRET systems in conjunction with GFP derivatives like YFP as acceptor domains Advantages and disadvantages editThe main advantage of FbFPs over GFP is their independence of molecular oxygen Since all GFP derivatives and homologues require molecular oxygen for the maturation of their chromophore these fluorescent proteins are of limited use under anaerobic or hypoxic conditions 13 Since FbFPs bind FMN as chromophore which is synthesized independently of molecular oxygen their fluorescence signal does not differ between aerobic and anaerobic conditions 1 14 Another advantage is the small size of FbFPs which is typically between 100 and 150 amino acids This is about half the size of GFP 238 amino acids It could for example be shown that this renders them superior tags for monitoring tobacco mosaic virus infections in tobacco leaves 6 Due to their extraordinary long average fluorescence lifetime of up to 5 7 ns they are also very well suited for the use as donor domains in FRET systems in conjunction with e g YFP see photophysical properties A fusion of EcFbFP and YFP was e g used to develop the first genetically encoded fluorescence biosensor for oxygen FluBO 15 The main disadvantage compared to GFP variants is their lower brightness the product of e and F The commonly used EGFP e 55 000 M 1cm 1 F 0 60 16 for example is approximately five times as bright as EcFbFP Another disadvantage of the FbFPs is the lack of color variants to tag and distinguish multiple proteins in a single cell or tissue The largest spectral shift reported for FbFPs so far is 10 nm Although this variant Pp2FbFP Q116V can be visually distinguished from the others with the human eye 8 the spectral differences are too small for fluorescence microscopy filters References edit a b c Drepper T Eggert T Circolone F Heck A Krauss U Guterl JK Wendorff M Losi A Gartner W Jaeger KE 2007 Reporter proteins for in vivo fluorescence without oxygen Nat Biotechnol 25 4 443 445 doi 10 1038 nbt1293 PMID 17351616 S2CID 7335755 a b c Christie JM Reymond P Powell GK Bernasconi P Raibekas AA Liscum E Briggs WR 1998 Arabidopsis NPH1 a flavoprotein with the properties of a photoreceptor for phototropism Science 282 5394 1698 1701 Bibcode 1998Sci 282 1698C doi 10 1126 science 282 5394 1698 PMID 9831559 Circolone F Granzin J Jentzsch K Drepper T Jaeger KE Willbold D Krauss U Batra Safferling R 2012 Structural Basis for the Slow Dark Recovery of a Full Length LOV Protein from Pseudomonas putida J Mol Biol 417 4 362 374 doi 10 1016 j jmb 2012 01 056 PMID 22326872 Losi A Gartner W 2011 Old chromophores new photoactivation paradigms trendy applications flavins in LOV and BLUF photoreceptors Photochem Photobiol 87 3 491 510 doi 10 1111 j 1751 1097 2011 00913 x PMID 21352235 Crosson S Moffat K 2001 Structure of a flavin binding plant photoreceptor domain insights into light mediated signal transduction Proc Natl Acad Sci U S A 98 6 2995 3000 Bibcode 2001PNAS 98 2995C doi 10 1073 pnas 051520298 PMC 30595 PMID 11248020 a b c Chapman S Faulkner C Kaiserli E Garcia Mata C Savenkov EI Roberts AG Oparka KJ Christie JM 2008 The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection Proc Natl Acad Sci U S A 105 50 20038 20043 Bibcode 2008PNAS 10520038C doi 10 1073 pnas 0807551105 PMC 2604982 PMID 19060199 Mukherjee A Weyant KB Walker J Schroeder CM 2012 Directed evolution of bright mutants of an oxygen independent flavin binding fluorescent protein from Pseudomonas putida Journal of Biological Engineering 6 1 20 doi 10 1186 1754 1611 6 20 PMC 3488000 PMID 23095243 a b c d e f g h Wingen M Potzkei J Endres S Casini G Rupprecht C Fahlke C Krauss U Jaeger KE Drepper T Gensch T 2014 The photophysics of LOV based fluorescent proteins new tools for cell biology Photochem Photobiol Sci 13 6 875 883 doi 10 1039 c3pp50414j PMID 24500379 S2CID 6068541 Rollen Katrin Granzin Joachim Remeeva Alina Davari Mehdi D Gensch Thomas Nazarenko Vera V Kovalev Kirill Bogorodskiy Andrey Borshchevskiy Valentin Hemmer Stefanie Schwaneberg Ulrich Gordeliy Valentin Jaeger Karl Erich Batra Safferling Renu Gushchin Ivan Krauss Ulrich 2021 04 13 The molecular basis of spectral tuning in blue and red shifted flavin binding fluorescent proteins The Journal of Biological Chemistry 296 100662 doi 10 1016 j jbc 2021 100662 ISSN 1083 351X PMC 8131319 PMID 33862085 Whitby L G 1953 A new method for preparing flavin adenine dinucleotide Biochem J 54 3 437 442 doi 10 1042 bj0540437 PMC 1269010 PMID 13058921 Goedhart J von Stetten D Noirclerc Savoye M Lelimousin M Joosen L Hink MA van Weeren L Gadella TW Royant A 2012 Structure guided evolution of cyan fluorescent proteins towards a quantum yield of 93 Nat Commun 3 751 Bibcode 2012NatCo 3 751G doi 10 1038 ncomms1738 PMC 3316892 PMID 22434194 Jung G Brockhinke A Gensch T Hotzer B Schwedler S Veettil SK 2012 Fluorescent Proteins I From Understanding to Design Vol 11 Springer Berlin Heidelberg ISBN 978 3 642 23371 5 Drepper T Gensch T Pohl M 2013 Advanced in vivo applications of blue light photoreceptors as alternative fluorescent proteins Photochem Photobiol Sci 12 7 1125 34 doi 10 1039 c3pp50040c PMID 23660639 S2CID 39879469 Walter J Hausmann S Drepper T Puls M Eggert T Dihne M 2012 Flavin mononucleotide based fluorescent proteins function in mammalian cells without oxygen requirement PLOS ONE 7 9 e43921 Bibcode 2012PLoSO 743921W doi 10 1371 journal pone 0043921 PMC 3439463 PMID 22984451 Potzkei J Kunze M Drepper T Gensch T Jaeger KE Buchs J 2012 Real time determination of intracellular oxygen in bacteria using a genetically encoded FRET based biosensor BMC Biol 10 28 doi 10 1186 1741 7007 10 28 PMC 3364895 PMID 22439625 Patterson G Day RN Piston D 2001 Fluorescent protein spectra J Cell Sci 114 Pt 5 837 838 doi 10 1242 jcs 114 5 837 PMID 11181166 Retrieved from https en wikipedia org w index php title FMN binding fluorescent protein amp oldid 1188014810, wikipedia, wiki, book, books, library,

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