RoGFP explained

reduction-oxidation sensitive Green fluorescent protein (roGFP)
Width:400
Symbol:roGFP
Pdb:1JC1

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.[1]

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.[2]

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.[3]

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. The roGFP1-R12 variant has been used to monitor redox potential in bacteria and yeast,[4] [5] but also for studies of spatially-organized redox potential in live, multicellular organisms such as the model nematode C. elegans.[6] In addition, roGFPs are used to investigate the topology of ER proteins, or to analyze the ROS production capacity of chemicals.[7] [8]

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).[9] 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.[10]

Species of roGFP

See Kostyulk 2020 [11] for a more comprehensive review of different redox sensors.

Caption text
Name Analyte Citation
roGFP1-roGFP6 EGSH
roGFP1_Rx Family EGSH
roGFP1-iX Family EGSH[12]
Grx1-roGFP2 EGSH
Mrx1-roGFP2 EMSH[13]
Brx-roGFP2 EBSH[14]
Tpx-roGFP2 ET(SH)2[15]
Orp1-roGFP2 H2O2 [16]
roGFP2-Tsa2DCR H2O2 [17]

See also

Notes and References

  1. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ . Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators . The Journal of Biological Chemistry . 279 . 13 . 13044–53 . March 2004 . 14722062 . 10.1074/jbc.M312846200 . free .
  2. Schwarzländer M, Fricker MD, Müller C, Marty L, Brach T, Novak J, Sweetlove LJ, Hell R, Meyer AJ . 6 . Confocal imaging of glutathione redox potential in living plant cells . Journal of Microscopy . 231 . 2 . 299–316 . August 2008 . 18778428 . 10.1111/j.1365-2818.2008.02030.x . amp . 28455264 .
  3. Meyer AJ, Brach T, Marty L, Kreye S, Rouhier N, Jacquot JP, Hell R . 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 . December 2007 . 17892447 . 10.1111/j.1365-313X.2007.03280.x . amp . free .
  4. Liu J, Wang Z, Kandasamy V, Lee SY, Solem C, Jensen PR . Harnessing the respiration machinery for high-yield production of chemicals in metabolically engineered Lactococcus lactis . Metabolic Engineering . 44 . 22–29 . November 2017 . 28890188 . 10.1016/j.ymben.2017.09.001 . 23405962 .
  5. Yu S, Qin W, Zhuang G, Zhang X, Chen G, Liu W . 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 . May 2009 . 19184609 . 10.1007/s00284-008-9354-y .
  6. Romero-Aristizabal C, Marks DS, Fontana W, Apfeld J . Regulated spatial organization and sensitivity of cytosolic protein oxidation in Caenorhabditis elegans . Nature Communications . 5 . 1 . 5020 . September 2014 . 25262602 . 10.1038/ncomms6020 . 4181376 . 2014NatCo...5.5020R .
  7. Brach T, Soyk S, Müller C, Hinz G, Hell R, Brandizzi F, Meyer AJ . Non-invasive topology analysis of membrane proteins in the secretory pathway . The Plant Journal . 57 . 3 . 534–41 . February 2009 . 18939964 . 10.1111/j.1365-313X.2008.03704.x .
  8. Schwarzländer M, Fricker MD, Sweetlove LJ . 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 . May 2009 . 19366606 . 10.1016/j.bbabio.2009.01.020 .
  9. Gutscher M, Pauleau AL, Marty L, Brach T, Wabnitz GH, Samstag Y, Meyer AJ, Dick TP . 6 . Real-time imaging of the intracellular glutathione redox potential . Nature Methods . 5 . 6 . 553–9 . June 2008 . 18469822 . 10.1038/NMETH.1212 . amp . 8947388 .
  10. Stanley JA, Johnsen SB, Apfeld J . The SensorOverlord predicts the accuracy of measurements with ratiometric biosensors . Scientific Reports . 10 . 1 . 16843 . October 2020 . 33033364 . 10.1038/s41598-020-73987-0 . 7544824 . 2020NatSR..1016843S .
  11. Kostyuk AI, Panova AS, Kokova AD, Kotova DA, Maltsev DI, Podgorny OV, Belousov VV, Bilan DS . 6 . In Vivo Imaging with Genetically Encoded Redox Biosensors . International Journal of Molecular Sciences . 21 . 21 . 8164 . October 2020 . 33142884 . 10.3390/ijms21218164 . 7662651 . free .
  12. Lohman JR, Remington SJ . Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments . Biochemistry . 47 . 33 . 8678–88 . August 2008 . 18652491 . 10.1021/bi800498g .
  13. Bhaskar A, Chawla M, Mehta M, Parikh P, Chandra P, Bhave D, Kumar D, Carroll KS, Singh A . 6 . Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection . PLOS Pathogens . 10 . 1 . e1003902 . January 2014 . 24497832 . 10.1371/journal.ppat.1003902 . Christopher M. Sassetti (ed.) . 3907381 . free .
  14. Loi VV, Harms M, Müller M, Huyen NT, Hamilton CJ, Hochgräfe F, Pané-Farré J, Antelmann H . 6 . 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 . May 2017 . 27462976 . 10.1089/ars.2016.6733 . 5444506 .
  15. Ebersoll S, Bogacz M, Günter LM, Dick TP, Krauth-Siegel RL . A tryparedoxin-coupled biosensor reveals a mitochondrial trypanothione metabolism in trypanosomes . eLife . 9 . –53227 . January 2020 . 32003744 . 10.7554/eLife.53227 . 7046469 . free .
  16. Gutscher M, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, Samstag Y, Dick TP . Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases . The Journal of Biological Chemistry . 284 . 46 . 31532–40 . November 2009 . 19755417 . 10.1074/jbc.M109.059246 . 2797222 . free .
  17. Morgan B, Van Laer K, Owusu TN, Ezeriņa D, Pastor-Flores D, Amponsah PS, Tursch A, Dick TP . 6 . Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes . Nature Chemical Biology . 12 . 6 . 437–43 . June 2016 . 27089028 . 10.1038/nchembio.2067 .

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