FMN-binding fluorescent proteins

from Wikipedia, the free encyclopedia
FMN-binding fluorescent proteins
FMN-binding fluorescent proteins
Typical core domain of an FbFP shown using the example of PDB  2PR5
Mass / length primary structure approx. 100-150 amino acids / approx. 11-16 kDa
Secondary to quaternary structure Monomer or dimer
Cofactor FMN

FMN-binding fluorescent proteins ( English FMN-binding fluorescent proteins , FbFP) are a class of oxygen-independent fluorescent proteins , the flavin (FMN) as a chromophore binding.

General

FbFPs were developed from blue light receptors (the so-called light-oxygen-voltage-sensing domains , LOV domains ) such as those found in plants and bacteria. They complement the fluorescent proteins of the GFP derivatives and homologues and are characterized in particular by their oxygen independence and their small size. They absorb blue light and emit light in the cyan-green spectral range .

development

LOV domains are a subclass of the PAS domains and were first discovered in plants as part of the phototropin , which plays a central role in the plant's growth towards light. They bind flavin mononucleotide (FMN) non-covalently as a cofactor , which is required for the absorption of blue light. Because of the bound FMN, LOV domains have inherent fluorescence, which is, however, very weak. When irradiated with blue light, LOV domains also undergo a photocycle in which a covalent bond is formed between a conserved cysteine ​​residue and the FMN. This leads to a change in conformation of the protein for signal transmission and to a loss of fluorescence. The covalent bond is energetically unfavorable and is split again within seconds to hours, depending on the protein. In order to make better use of the fluorescence properties of these proteins, the natural photocycle of the proteins was switched off by genetic modification by replacing the conserved cysteine ​​with an alanine . As a result, the protein remains in the fluorescent state even when irradiated with blue light and the fluorescence intensity is significantly increased.

The first FbFPs could be further improved by various mutagenesis methods. In particular, the brightness but also the photostability of the proteins was improved and their spectral properties changed.

Spectral properties

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

FbFPs have an excitation maximum at a wavelength of approx. 450 nm (blue light) and a second pronounced excitation peak at approx. 370 nm ( UV-A light ). The emission maximum is approx. 495 nm, with a shoulder at approx. 520 nm. There is also a variant of Pp2FbFP (Q116V) whose excitation and emission spectrum is shifted by 10 nm towards shorter wavelengths.

Photophysical properties

The photophysical properties of the FbFPs are determined by the chromophore and its chemical environment in the protein. The extinction coefficient (ε) for all known FbFPs is around 14,200 M −1 cm −1 and is thus somewhat higher than that of free FMN (ε = 12,200 M −1 cm −1 .) The fluorescence quantum yield (Φ) varies greatly between the different FbFPs and ranges from 0.2 (phiLOV2.1) to 0.44 (EcFbFP and iLOV). This means that the fluorescence quantum yield of free FMN (Φ = 0.25) when bound to the protein is almost doubled in some cases. An even clearer difference can be seen in the photostability, the resistance of the fluorescent proteins to bleaching in the event of prolonged, strong exposure to blue light. For example, with the phiLOV2.1, which is optimized for photostability, it takes around 40 times as long for half of the fluorescence to fade than with free FMN. This stabilizing effect can be observed in almost all FbFPs, but it is usually only around 5 to 10 times. The average fluorescence lifetime of the individual FbFPs is between 3.17 ns (Pp2FbFP) and 5.7 ns (e.g. EcFbFP). They are therefore significantly longer than the typical lifetimes of GFP derivatives, which are typically between 1.5 and 3 ns. FbFPs are therefore well suited as donor domains in Förster resonance energy transfer (FRET) systems in conjunction with GFP derivatives such as. B. YFP.

Advantages and disadvantages

The greatest advantage of FbFPs over GFP is their independence from molecular oxygen. Since GFP and all its derivatives and homologues require molecular oxygen for the maturation of the chromophore, these fluorescent proteins can only be used to a limited extent under hypoxic and anaerobic conditions. Since FbFPs bind FMN as a cofactor, whose synthesis does not require molecular oxygen, the fluorescence signal of these proteins is the same under aerobic and anaerobic conditions. FbFPs are typically between 100 and 150 amino acids long and therefore only about half the size of GFP (238 amino acids). It could be shown that this small size z. B. in the marking of the tobacco mosaic virus is advantageous. Due to the long fluorescence lifetime of up to 5.7 ns, FbFPs are well suited as donors for FRET systems in connection with GFP derivatives such as e.g. B. YFP. A fusion of EcFbFP and YFP has already been used to develop the first genetically encoded fluorescence biosensor for oxygen (FluBO).

The main disadvantage of FbFPs compared to GFP is their lower brightness (the product of ε and Φ). The frequently used EGFP with ε = 55,000 M −1 cm −1 and Φ = 0.60 is about five times as bright as EcFbFP. Another disadvantage of FbFPs is the lack of color variants to date to mark several proteins within a cell or tissue and to differentiate them under the microscope. The largest spectral shift to date in FbFP variants is only 10 nm. This means that the protein can be distinguished from others with the human eye, but this shift is not sufficient for a differentiation using fluorescence filters such as those used in microscopes.

Individual evidence

  1. ↑ a b c Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauss, U., Guterl, JK, Wendorff, M., Losi, A., Gärtner, W., Jaeger, KE: Reporter proteins for in vivo fluorescence without oxygen . In: Nat Biotechnol . 25, 2007, pp. 443-445. doi : 10.1038 / nbt1293 . PMID 17351616 .
  2. ↑ a b Christie, JM, Reymond, P., Powell, GK, Bernasconi, P., Raibekas, AA, Liscum, E., Briggs, WR: Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism . In: Science . 282, 1998, pp. 1698-1701. 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 .: Structural Basis for the Slow Dark Recovery of a full-length LOV protein from Pseudomonas putida . In: J Mol Biol . 417, 2012, pp. 362-374. doi : 10.1016 / j.jmb.2012.01.056 . PMID 22326872 .
  4. ↑ Losi, A. & Gärtner, W .: Old chromophores, new photoactivation paradigms, trendy applications: flavins in LOV and BLUF photoreceptors. . In: Photochem Photobiol . 87, 2011, pp. 491-510. doi : 10.1111 / j.1751-1097.2011.00913.x . PMID 21352235 .
  5. ↑ Crosson, S. & Moffat, K .: Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction . In: Proc Natl Acad Sci USA . 98, 2001, pp. 2995-3000. doi : 10.1073 / pnas.051520298 . PMID 11248020 .
  6. ↑ a b c Chapman, S., Faulkner, C., Kaiserli, E., Garcia-Mata, C., Savenkov, EI, Roberts, AG, Oparka, KJ, Christie, JM: The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection . In: Proc Natl Acad Sci USA . 105, 2008, pp. 20038-20043. doi : 10.1073 / pnas.0807551105 . PMID 19060199 .
  7. ↑ Mukherjee, A., Weyant, KB, Walker, J. & Schroeder, CM: Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida . In: Journal of Biological Engineering . 6, 2012, p. 20. doi : 10.1186 / 1754-1611-6-20 . 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 .: The photophysics of LOV-based fluorescent proteins - new tools for cell biology . In: Photochem Photobiol Sci . 13, 2014, pp. 875-883. doi : 10.1039 / c3pp50414j . PMID 24500379 .
  9. ^ Whitby, LG: A new method for preparing flavin-adenine dinucleotide . In: Biochem J . 54, 1953, pp. 437-442. PMID 13058921 .
  10. ↑ Goedhart, J., von Stetten, D., Noirclerc-Savoye, M., Lelimousin, M., Joosen, L., Hink, MA, van Weeren, L., Gadella, TW, Jr., Royant, A. : Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93% . In: Nat Commun . 3, 2012, p. 751. doi : 10.1038 / ncomms1738 . PMID 22434194 .
  11. ↑ Jung, G., Brockhinke, A., Gensch, T., Hötzer, B., Schwedler, S., Veettil, SK: Fluorescent Proteins I. From Understanding to Design Vol.11 . Springer Berlin Heidelberg, 2012, ISBN 978-3-642-23371-5 .
  12. ↑ Drepper, T., Gensch, T., Pohl, M .: Advanced in vivo applications of blue light photoreceptors as alternative fluorescent proteins . In: Photochem Photobiol Sci . 12, 2013, pp. 1125-1134. doi : 10.1039 / c3pp50040c . PMID 23660639 .
  13. ↑ Walter, J., Hausmann, S., Drepper, T., Puls, M., Eggert, T., Dihné, M: Flavin mononucleotide-based fluorescent proteins function in mammalian cells without oxygen requirement . In: PLoS One . 7, 2012, p. E43921. doi : 10.1371 / journal.pone.0043921 . PMID 22984451 .
  14. ↑ Potzkei, J., Kunze, M., Drepper, T., Gensch, T., Jaeger, KE, Büchs, J .: Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor . In: BMC Biol . 10, 2012, p. 28. doi : 10.1186 / 1741-7007-10-2 . PMID 22439625 .
  15. ↑ Patterson, G., Day, RN & Piston, D .: Fluorescent protein spectra . In: J Cell Sci . 114, 2001, pp. 837-838. PMID 11181166 .