RoGFP

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reduction-oxidation sensitive Green fluorescent protein (roGFP)
The oxidized and reduced forms of the redox-sensitive Green Fluorescent Protein 1-R7 (roGFP1-R7).[1]
Identifiers
SymbolroGFP
PDB1JC1

The reduction-oxidation sensitive green fluorescent protein (roGFP) is a green fluorescent protein engineered to be sensitive to changes in the local redox environment. roGFPs are used as redox-sensitive biosensors.

In 2004, researchers in S. James Remington's lab at the University of Oregon constructed the first roGFPs by introducing two cysteines into the beta barrel structure of GFP. The resulting engineered protein could exist in two different oxidation states (reduced dithiol or oxidized disulfide), each with different fluorescent properties.[2]

Originally, members of the Remington lab published six versions of roGFP, termed roGFP1-6 (see more structural details below). Different groups of researchers introduced cysteines at different locations in the GFP molecule, generally finding that cysteines introduced at the amino acid positions 147 and 204 produced the most robust results.[3]

roGFPs are often genetically encoded into cells for in-vivo imaging of redox potential. In cells, roGFPs can generally be modified by redox enzymes such as glutaredoxin or thioredoxin. roGFP2 preferentially interacts with glutaredoxins and therefore reports the cellular glutathione redox potential.[4]

Various attempts have been made to make roGFPs that are more amenable to live-cell imaging. Most notably, substituting three positively-charged amino acids adjacent to the disulfide in roGFP1 drastically improves the response rate of roGFPs to physiologically relevant changes in redox potential. The resulting roGFP variants, named roGFP1-R1 through roGFP1-R14, are much more suitable for live-cell imaging.[1] The roGFP1-R12 variant has been used to monitor redox potential in bacteria and yeast,[5][6] but also for studies of spatially-organized redox potential in live, multicellular organisms such as the model nematode C. elegans.[7] In addition, roGFPs are used to investigate the topology of ER proteins, or to analyze the ROS production capacity of chemicals.[8] [9]

One notable improvement to roGFPs occurred in 2008, when the specificity of roGFP2 for glutathione was further increased by linking it to the human glutaredoxin 1 (Grx1).[10] By expressing the Grx1-roGFP fusion sensors in the organism of interest and/or targeting the protein to a cellular compartment, it is possible to measure the glutathione redox potential in a specific cellular compartment in real-time and therefore provides major advantages compared to other invasive static methods e.g. HPLC.

Given the variety of roGFPs, some effort has been made to benchmark their performance. For example, members of Javier Apfeld's group published a method in 2020 describing the 'suitable ranges' of different roGFPs, determined by how sensitive each sensor is to experimental noise in different redox conditions.[11]

Species of roGFP

See Kostyulk 2020 [12] for a more comprehensive review of different redox sensors.

Caption text
Name Analyte Citation
roGFP1-roGFP6 EGSH [2]
roGFP1_Rx Family EGSH [1]
roGFP1-iX Family EGSH [13]
Grx1-roGFP2 EGSH [10]
Mrx1-roGFP2 EMSH [14]
Brx-roGFP2 EBSH [15]
Tpx-roGFP2 ET(SH)2 [16]
Orp1-roGFP2 H2O2 [17]
roGFP2-Tsa2DCR H2O2 [18]

See also

References

  1. ^ a b c Cannon MB, Remington SJ (January 2006). "Re-engineering redox-sensitive green fluorescent protein for improved response rate". Protein Science. 15 (1): 45–57. doi:10.1110/ps.051734306. PMC 2242357. PMID 16322566.
  2. ^ a b Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ (March 2004). "Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators". The Journal of Biological Chemistry. 279 (13): 13044–53. doi:10.1074/jbc.M312846200. PMID 14722062.
  3. ^ Schwarzländer M, Fricker MD, Müller C, Marty L, Brach T, Novak J, et al. (August 2008). "Confocal imaging of glutathione redox potential in living plant cells". Journal of Microscopy. 231 (2): 299–316. doi:10.1111/j.1365-2818.2008.02030.x. PMID 18778428. S2CID 28455264.
  4. ^ Meyer AJ, Brach T, Marty L, Kreye S, Rouhier N, Jacquot JP, Hell R (December 2007). "Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer". The Plant Journal. 52 (5): 973–86. doi:10.1111/j.1365-313X.2007.03280.x. PMID 17892447.
  5. ^ Liu J, Wang Z, Kandasamy V, Lee SY, Solem C, Jensen PR (November 2017). "Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis" (PDF). Metabolic Engineering. 44: 22–29. doi:10.1016/j.ymben.2017.09.001. PMID 28890188. S2CID 23405962.
  6. ^ Yu S, Qin W, Zhuang G, Zhang X, Chen G, Liu W (May 2009). "Monitoring oxidative stress and DNA damage induced by heavy metals in yeast expressing a redox-sensitive green fluorescent protein". Current Microbiology. 58 (5): 504–10. doi:10.1007/s00284-008-9354-y. PMID 19184609.
  7. ^ Romero-Aristizabal C, Marks DS, Fontana W, Apfeld J (September 2014). "Regulated spatial organization and sensitivity of cytosolic protein oxidation in Caenorhabditis elegans". Nature Communications. 5 (1): 5020. Bibcode:2014NatCo...5.5020R. doi:10.1038/ncomms6020. PMC 4181376. PMID 25262602.
  8. ^ Brach T, Soyk S, Müller C, Hinz G, Hell R, Brandizzi F, Meyer AJ (February 2009). "Non-invasive topology analysis of membrane proteins in the secretory pathway". The Plant Journal. 57 (3): 534–41. doi:10.1111/j.1365-313X.2008.03704.x. PMID 18939964.
  9. ^ Schwarzländer M, Fricker MD, Sweetlove LJ (May 2009). "Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1787 (5): 468–75. doi:10.1016/j.bbabio.2009.01.020. PMID 19366606.
  10. ^ a b Gutscher M, Pauleau AL, Marty L, Brach T, Wabnitz GH, Samstag Y, et al. (June 2008). "Real-time imaging of the intracellular glutathione redox potential". Nature Methods. 5 (6): 553–9. doi:10.1038/NMETH.1212. PMID 18469822. S2CID 8947388.
  11. ^ Stanley JA, Johnsen SB, Apfeld J (October 2020). "The SensorOverlord predicts the accuracy of measurements with ratiometric biosensors". Scientific Reports. 10 (1): 16843. Bibcode:2020NatSR..1016843S. doi:10.1038/s41598-020-73987-0. PMC 7544824. PMID 33033364.
  12. ^ Kostyuk AI, Panova AS, Kokova AD, Kotova DA, Maltsev DI, Podgorny OV, et al. (October 2020). "In Vivo Imaging with Genetically Encoded Redox Biosensors". International Journal of Molecular Sciences. 21 (21): 8164. doi:10.3390/ijms21218164. PMC 7662651. PMID 33142884.
  13. ^ Lohman JR, Remington SJ (August 2008). "Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments". Biochemistry. 47 (33): 8678–88. doi:10.1021/bi800498g. PMID 18652491.
  14. ^ Bhaskar A, Chawla M, Mehta M, Parikh P, Chandra P, Bhave D, et al. (January 2014). "Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection". PLOS Pathogens. 10 (1). Christopher M. Sassetti (ed.): e1003902. doi:10.1371/journal.ppat.1003902. PMC 3907381. PMID 24497832.
  15. ^ Loi VV, Harms M, Müller M, Huyen NT, Hamilton CJ, Hochgräfe F, et al. (May 2017). "Real-Time Imaging of the Bacillithiol Redox Potential in the Human Pathogen Staphylococcus aureus Using a Genetically Encoded Bacilliredoxin-Fused Redox Biosensor". Antioxidants & Redox Signaling. 26 (15): 835–848. doi:10.1089/ars.2016.6733. PMC 5444506. PMID 27462976.
  16. ^ Ebersoll S, Bogacz M, Günter LM, Dick TP, Krauth-Siegel RL (January 2020). "A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes". eLife. 9: –53227. doi:10.7554/eLife.53227. PMC 7046469. PMID 32003744.
  17. ^ Gutscher M, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, Samstag Y, Dick TP (November 2009). "Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases". The Journal of Biological Chemistry. 284 (46): 31532–40. doi:10.1074/jbc.M109.059246. PMC 2797222. PMID 19755417.
  18. ^ Morgan B, Van Laer K, Owusu TN, Ezeriņa D, Pastor-Flores D, Amponsah PS, et al. (June 2016). "Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes". Nature Chemical Biology. 12 (6): 437–43. doi:10.1038/nchembio.2067. PMID 27089028.
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