Photoconversion of the Green Fluorescent Protein and Related Proteins

  • Jasper J. van ThorEmail author
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)


This review focuses on the mechanistic details of photochromic reactions of the green fluorescent protein (GFP) and also of its mutant derivatives and related fluorescent proteins. A number of distinct photochromic processes have so far been identified that have entirely different photochemical and chemical basis, which will be reviewed. In addition to bright fluorescence, the GFP from the jellyfish Aequorea victoria undergoes photochromic transformation with blue or UV illumination. The associated change in electronic absorption provides a spectroscopic contrast that can be used in fluorescence microscopy application to tag and track the movement of populations that are photoconverted. Key to the successful use of photoconversion for such microscopy experiments is in fact the relatively low quantum yield of the irreversible process. In the wild-type GFP, photoconversion is triggered by light-induced electron transfer from the buried anionic carboxylate of Glu222 to the optically excited protonated chromophore. An unstable carboxylate radical subsequently cleaves off a CO2 molecule in a “Kolbe” type reaction that has been trapped in a partially oriented site near the chromophore-binding site at 100K, as observed by low-temperature X-ray crystallography and cryo-infrared crystallography. Structural intermediates in the subsequent relaxation pathway involve motion of CO2, amino acids and H-bonded waters both in the chromophore vicinity and at longer range. This review provides an overview of the molecular characterisation using structural and spectroscopy methods of this photoconversion reaction of GFP. In addition, the mechanisms of photochromic reactions of mutants of GFP and related fluorescent proteins will be summarised and discussed. These include the cistrans isomerisation and protonation changes in Dronpa, asFP595 and IrisFP and related proteins, light-induced maturation in aceGFPL, and photoinduced beta-elimination and backbone cleavage that leads to “green-to-red” photoconversion in EosFP, Kaede, IrisFP and KikGR.


asFP595 Dronpa Fluorescent proteins GFP Green fluorescent protein ‘Green-to-red’ photoconversion Kaede Oxidative decarboxylation Photoconversion Photoisomerisation Photoswitching 



Jasper van Thor is a Royal Society University Research Fellow. JvT acknowledges support from the European Research Council (Grant Agreement N° 208650) and EPSRC (Grant Ref EP/I003304/1).


  1. 1.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805Google Scholar
  2. 2.
    Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233Google Scholar
  3. 3.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544Google Scholar
  4. 4.
    Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395Google Scholar
  5. 5.
    Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ (1997) Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 94:2306–2311Google Scholar
  6. 6.
    Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251Google Scholar
  7. 7.
    Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455Google Scholar
  8. 8.
    Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664Google Scholar
  9. 9.
    Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91:12501–12504Google Scholar
  10. 10. (2008) The Nobel Prize in Chemistry 2008 – Press Release. “for the discovery and development of the green fluorescent protein, GFP”.,
  11. 11.
    Morise H, Shimomura O, Johnson FH, Winant J (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 13:2656–2662Google Scholar
  12. 12.
    Perozzo MA, Ward KB, Thompson RB, Ward WW (1988) X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals. J Biol Chem 263:7713–7716Google Scholar
  13. 13.
    Scharnagl C, Raupp-Kossmann R, Fischer SF (1999) Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations. Biophys J 77:1839–1857Google Scholar
  14. 14.
    Bokman SH, Ward WW (1981) Renaturation of Aequorea gree-fluorescent protein. Biochem Biophys Res Commun 101:1372–1380Google Scholar
  15. 15.
    Ward WW, Cody CW, Hart RC, Cormier MJ (1980) Spectrophotometric identity of the energy transfer chromophores in Renilla and Aequorea green-fluorescent proteins. Photochem Photobiol 31:611–615Google Scholar
  16. 16.
    Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci USA 93:8362–8367Google Scholar
  17. 17.
    Förster T (1949) Fluorezenzspektrum und Wasserstoffionenkonzentration. Naturwiss 36:186Google Scholar
  18. 18.
    Förster T (1950) Die pH-abhangigkeit der fluoreszenz von naphthalinderivaten. Z Electrochem 54:531Google Scholar
  19. 19.
    van Thor JJ, Gensch T, Hellingwerf KJ, Johnson LN (2002) Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat Struct Biol 9:37–41Google Scholar
  20. 20.
    van Thor JJ, Pierik AJ, Nugteren-Roodzant I, Xie A, Hellingwerf KJ (1998) Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy. Biochemistry 37:16915–16921Google Scholar
  21. 21.
    Lill MA, Helms V (2002) Proton shuttle in green fluorescent protein studied by dynamic simulations. Proc Natl Acad Sci USA 99:2778–2781Google Scholar
  22. 22.
    van Thor JJ (2009) Photoreactions and dynamics of the green fluorescent protein. Chem Soc Rev 38:2935–2950Google Scholar
  23. 23.
    Vendrell O, Gelabert R, Moreno M, Lluch JM (2008) Operation of the proton wire in green fluorescent protein. A quantum dynamics simulation. J Phys Chem B 112:5500–5511Google Scholar
  24. 24.
    Palm GJ, Zdanov A, Gaitanaris GA, Stauber R, Pavlakis GN, Wlodawer A (1997) The structural basis for spectral variations in green fluorescent protein. Nat Struct Biol 4:361–365Google Scholar
  25. 25.
    Stoner-Ma D, Jaye AA, Matousek P, Towrie M, Meech SR, Tonge PJ (2005) Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy. J Am Chem Soc 127:2864–2865Google Scholar
  26. 26.
    van Thor JJ, Georgiev GY, Towrie M, Sage JT (2005) Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein. J Biol Chem 280:33652–33659Google Scholar
  27. 27.
    van Thor JJ, Ronayne KL, Towrie M, Sage JT (2008) Balance between ultrafast parallel reactions in the green fluorescent protein has a structural origin. Biophys J 95:1902–1912Google Scholar
  28. 28.
    van Thor JJ, Zanetti G, Ronayne KL, Towrie M (2005) Structural events in the photocycle of green fluorescent protein. J Phys Chem B 109:16099–16108Google Scholar
  29. 29.
    Stoner-Ma D, Melief EH, Nappa J, Ronayne KL, Tonge PJ, Meech SR (2006) Proton relay reaction in green fluorescent protein (GFP): polarization-resolved ultrafast vibrational spectroscopy of isotopically edited GFP. J Phys Chem B 110:22009–22018Google Scholar
  30. 30.
    Youvan DC, Michel-Beyerle ME (1996) Structure and fluorescence mechanism of GFP. Nat Biotechnol 14:1219–1220Google Scholar
  31. 31.
    Henderson JN, Osborn MF, Koon N, Gepshtein R, Huppert D, Remington SJ (2009) Excited state proton transfer in the red fluorescent protein mKeima. J Am Chem Soc 131(37):13212–13213Google Scholar
  32. 32.
    Piatkevich KD, Malashkevich VN, Almo SC, Verkhusha VV (2010) Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large stokes shift. J Am Chem Soc 132:10762–10770Google Scholar
  33. 33.
    Lippincott-Schwartz J, Manley S (2009) Putting super-resolution fluorescence microscopy to work. Nat Methods 6:21–23Google Scholar
  34. 34.
    Lippincott-Schwartz J, Patterson GH (2008) Fluorescent proteins for photoactivation experiments. Methods Cell Biol 85:45–61Google Scholar
  35. 35.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877Google Scholar
  36. 36.
    van Thor JJ, Sage JT (2006) Charge transfer in green fluorescent protein. Photochem Photobiol Sci 5:597–602Google Scholar
  37. 37.
    Bell AF, Stoner-Ma D, Wachter RM, Tonge PJ (2003) Light-driven decarboxylation of wild-type green fluorescent protein. J Am Chem Soc 125:6919–6926Google Scholar
  38. 38.
    Schneider M, Barozzi S, Testa I, Faretta M, Diaspro A (2005) Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region. Biophys J 89:1346–1352Google Scholar
  39. 39.
    Langhojer F, Dimler F, Jung G, Brixner T (2009) Ultrafast photoconversion of the green fluorescent protein studied by accumulative femtosecond spectroscopy. Biophys J 96:2763–2770Google Scholar
  40. 40.
    Habuchi S, Cotlet M, Gensch T, Bednarz T, Haber-Pohlmeier S, Rozenski J, Dirix G, Michiels J, Vanderleyden J, Heberle J, De Schryver FC, Hofkens J (2005) Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. J Am Chem Soc 127:8977–8984Google Scholar
  41. 41.
    He X, Bell AF, Tonge P (2002) Isotopic labeling and normal-mode analysis of a model green fluorescent protein chromophore. J Phys Chem B 106:6056–6066Google Scholar
  42. 42.
    Bublitz GU, Boxer SG (1997) Stark spectroscopy: applications in chemistry, biology, and materials science. Annu Rev Phys Chem 48:213–242Google Scholar
  43. 43.
    Anderson JM, Kocji JK (1970) Manganese(III) complexes in oxidative decarboxylation of acids. J Am Chem Soc 92:2450–2460Google Scholar
  44. 44.
    Kolbe H (1849) Untersuchungen über die Elektrolyse organischer Verbindungen. Ann Chem Pharm 69:257–294Google Scholar
  45. 45.
    Cao W, Ye X, Sjodin T, Christian JF, Demidov AA, Berezhna S, Wang W, Barrick D, Sage JT, Champion PM (2004) Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements. Biochemistry 43:11109–11117Google Scholar
  46. 46.
    Ye X, Yu A, Georgiev GY, Gruia F, Ionascu D, Cao W, Sage JT, Champion PM (2005) CO rebinding to protoheme: investigations of the proximal and distal contributions to the geminate rebinding barrier. J Am Chem Soc 127:5854–5861Google Scholar
  47. 47.
    Zeng W, Silvernail NJ, Wharton DC, Georgiev GY, Leu BM, Scheidt WR, Zhao J, Sturhahn W, Alp EE, Sage JT (2005) Direct probe of iron vibrations elucidates NO activation of heme proteins. J Am Chem Soc 127:11200–11201Google Scholar
  48. 48.
    Lossau H, Kummer A, Heinecke R, Pollinger-Dammer F, Kompa C, Bieser G, Jonsson T, Silva CM, Yang MM, Youvan DC, Michel-Beyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore-protein interactions. Chem Phys 213:1–16Google Scholar
  49. 49.
    Hopfield JJ (1974) Electron transfer between biological molecules by thermally activated tunneling. Proc Natl Acad Sci USA 71:3640–3644Google Scholar
  50. 50.
    Marcus RA, Sutin N (1985) Electron transfers in chemistry and biology. Biochim Biophys Acta 811:265–322Google Scholar
  51. 51.
    Moser CC, Keske JM, Warncke K, Farid RS, Dutton PL (1992) Nature of biological electron transfer. Nature 355:796–802Google Scholar
  52. 52.
    Page CC, Moser CC, Chen X, Dutton PL (1999) Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature 402:47–52Google Scholar
  53. 53.
    McAnaney TB, Zeng W, Doe CF, Bhanji N, Wakelin S, Pearson DS, Abbyad P, Shi X, Boxer SG, Bagshaw CR (2005) Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism. Biochemistry 44:5510–5524Google Scholar
  54. 54.
    Henderson JN, Gepshtein R, Heenan JR, Kallio K, Huppert D, Remington SJ (2009) Structure and mechanism of the photoactivatable green fluorescent protein. J Am Chem Soc 131:4176–4177Google Scholar
  55. 55.
    Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol Suppl:S7–S14Google Scholar
  56. 56.
    Lippincott-Schwartz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91Google Scholar
  57. 57.
    Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc 2:2024–2032Google Scholar
  58. 58.
    Chudakov DM, Lukyanov S, Lukyanov KA (2007b) Using photoactivatable fluorescent protein Dendra2 to track protein movement. Biotechniques 42:553, 555, 557 passimGoogle Scholar
  59. 59.
    Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA (2004) Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 22:1435–1439Google Scholar
  60. 60.
    Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, Zorov DB, Lukyanov S, Lukyanov KA (2003) Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol 21:191–194Google Scholar
  61. 61.
    Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005) Innovation: photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891Google Scholar
  62. 62.
    Verkhusha VV, Sorkin A (2005) Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem Biol 12:279–285Google Scholar
  63. 63.
    Schäfer LV, Groenhof G, Boggio-Pasqua M, Robb MA, Grubmuller H (2008) Chromophore protonation state controls photoswitching of the fluoroprotein asFP595. PLoS Comput Biol 4(3):e1000034Google Scholar
  64. 64.
    Schäfer LV, Groenhof G, Klingen AR, Ullmann GM, Boggio-Pasqua M, Robb MA, Grubmuller H (2007) Photoswitching of the fluorescent protein asFP595: mechanism, proton pathways, and absorption spectra. Angew Chem Int Ed Engl 46:530–536Google Scholar
  65. 65.
    Ando R, Hama H, Yamamoto-Hino M, Mizuno H, Miyawaki A (2002) An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc Natl Acad Sci USA 99:12651–12656Google Scholar
  66. 66.
    Nienhaus K, Nienhaus GU, Wiedenmann J, Nar H (2005) Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP. Proc Natl Acad Sci USA 102:9156–9159Google Scholar
  67. 67.
    Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101:15905–15910Google Scholar
  68. 68.
    Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS ONE 3(12):e3944Google Scholar
  69. 69.
    Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A (2005) Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep 6:233–238Google Scholar
  70. 70.
    Gurskaya NG, Verkhusha VV, Shcheglov AS, Staroverov DB, Chepurnykh TV, Fradkov AF, Lukyanov S, Lukyanov KA (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465Google Scholar
  71. 71.
    Tsutsui H, Shimizu H, Mizuno H, Nukina N, Furuta T, Miyawaki A (2009) The E1 mechanism in photo-induced beta-elimination reactions for green-to-red conversion of fluorescent proteins. Chem Biol 16:1140–1147Google Scholar
  72. 72.
    Mizuno H, Mal TK, Tong KI, Ando R, Furuta T, Ikura M, Miyawaki A (2003) Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell 12:1051–1058Google Scholar
  73. 73.
    Hayashi I, Mizuno H, Tong KI, Furuta T, Tanaka F, Yoshimura M, Miyawaki A, Ikura M (2007) Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede. J Mol Biol 372:918–926Google Scholar
  74. 74.
    Lelimousin M, Adam V, Nienhaus GU, Bourgeois D, Field MJ (2009) Photoconversion of the fluorescent protein EosFP: a hybrid potential simulation study reveals intersystem crossings. J Am Chem Soc 131:16814–16823Google Scholar
  75. 75.
    Gurskaya NG, Fradkov AF, Pounkova NI, Staroverov DB, Bulina ME, Yanushevich YG, Labas YA, Lukyanov S, Lukyanov KA (2003) A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem J 373:403–408Google Scholar
  76. 76.
    Pletneva NV, Pletnev VZ, Lukyanov KA, Gurskaya NG, Goryacheva EA, Martynov VI, Wlodawer A, Dauter Z, Pletnev S (2010) Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis. J Biol Chem 285:15978–15984Google Scholar
  77. 77.
    Barondeau DP, Tainer JA, Getzoff ED (2006) Structural evidence for an enolate intermediate in GFP fluorophore biosynthesis. J Am Chem Soc 128:3166–3168Google Scholar
  78. 78.
    Pouwels LJ, Zhang L, Chan NH, Dorrestein PC, Wachter RM (2008) Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants. Biochemistry 47:10111–10122Google Scholar
  79. 79.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373Google Scholar
  80. 80.
    Andresen M, Stiel AC, Trowitzsch S, Weber G, Eggeling C, Wahl MC, Hell SW, Jakobs S (2007) Structural basis for reversible photoswitching in Dronpa. Proc Natl Acad Sci USA 104:13005–13009Google Scholar
  81. 81.
    Stiel AC, Trowitzsch S, Weber G, Andresen M, Eggeling C, Hell SW, Jakobs S, Wahl MC (2007) 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem J 402:35–42Google Scholar
  82. 82.
    Andresen M, Wahl MC, Stiel AC, Grater F, Schafer LV, Trowitzsch S, Weber G, Eggeling C, Grubmuller H, Hell SW, Jakobs S (2005) Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci USA 102:13070–13074Google Scholar
  83. 83.
    Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP, Markelov ML, Zaraisky AG, Zhao X, Fang Y, Tan W, Lukyanov SA (2000) Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem 275:25879–25882Google Scholar
  84. 84.
    Quillin ML, Anstrom DM, Shu X, O’Leary S, Kallio K, Chudakov DM, Remington SJ (2005) Kindling fluorescent protein from Anemonia sulcata: dark-state structure at 1.38 A resolution. Biochemistry 44:5774–5787Google Scholar
  85. 85.
    Wilmann PG, Petersen J, Pettikiriarachchi A, Buckle AM, Smith SC, Olsen S, Perugini MA, Devenish RJ, Prescott M, Rossjohn J (2005) The 2.1A crystal structure of the far-red fluorescent protein HcRed: inherent conformational flexibility of the chromophore. J Mol Biol 349:223–237Google Scholar
  86. 86.
    Brakemann T, Weber G, Andresen M, Groenhof G, Stiel AC, Trowitzsch S, Eggeling C, Grubmuller H, Hell SW, Wahl MC, Jakobs S (2010) Molecular basis of the light-driven switching of the photochromic fluorescent protein Padron. J Biol Chem 285:14603–14609Google Scholar
  87. 87.
    Adam V, Lelimousin M, Boehme S, Desfonds G, Nienhaus K, Field MJ, Wiedenmann J, McSweeney S, Nienhaus GU, Bourgeois D (2008) Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc Natl Acad Sci USA 105:18343–18348Google Scholar
  88. 88.
    Faro AR, Adam V, Carpentier P, Darnault C, Bourgeois D, de Rosny E (2010) Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins. Photochem Photobiol Sci 9:254–262Google Scholar
  89. 89.
    Henderson JN, Ai HW, Campbell RE, Remington SJ (2007) Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc Natl Acad Sci USA 104:6672–6677Google Scholar
  90. 90.
    Li X, Chung LW, Mizuno H, Miyawaki A, Morokuma K (2010) A theoretical study on the nature of on- and off-states of reversibly photoswitching fluorescent protein Dronpa: absorption, emission, protonation, and Raman. J Phys Chem B 114:1114–1126Google Scholar
  91. 91.
    Mizuno H, Mal TK, Walchli M, Kikuchi A, Fukano T, Ando R, Jeyakanthan J, Taka J, Shiro Y, Ikura M, Miyawaki A (2008) Light-dependent regulation of structural flexibility in a photochromic fluorescent protein. Proc Natl Acad Sci USA 105:9227–9232Google Scholar
  92. 92.
    Habuchi S, Ando R, Dedecker P, Verheijen W, Mizuno H, Miyawaki A, Hofkens J (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci USA 102:9511–9516Google Scholar
  93. 93.
    Fron E, Flors C, Schweitzer G, Habuchi S, Mizuno H, Ando R, De Schryver FC, Miyawaki A, Hofkens J (2007) Ultrafast excited-state dynamics of the photoswitchable protein dronpa. J Am Chem Soc 129:4870–4871Google Scholar
  94. 94.
    Chudakov DM, Feofanov AV, Mudrik NN, Lukyanov S, Lukyanov KA (2003) Chromophore environment provides clue to “kindling fluorescent protein” riddle. J Biol Chem 278:7215–7219Google Scholar
  95. 95.
    Olsen S, Lamothe K, Martinez TJ (2010) Protonic gating of excited-state twisting and charge localization in GFP chromophores: a mechanistic hypothesis for reversible photoswitching. J Am Chem Soc 132:1192–1193Google Scholar
  96. 96.
    Polyakov IV, Grigorenko BL, Epifanovsky EM, Krylov AI, Nemukhin AV (2010) Potential energy landscape of the electronic states of the GFP chromophore in different protonation forms: electronic transition energies and conical intersections. J Chem Theory Comput 6:2377–2387Google Scholar
  97. 97.
    Bell AF, He X, Wachter RM, Tonge PJ (2000) Probing the ground state structure of the green fluorescent protein chromophore using Raman spectroscopy. Biochemistry 39:4423–4431Google Scholar
  98. 98.
    Schuttrigkeit TA, von Feilitzsch T, Kompa CK, Lukyanov KA, Savitsky AP, Voityuk AA, Michel-Beyerle ME (2006) Femtosecond study of light-induced fluorescence increase of the dark chromoprotein asFP595. Chem Phys 323:149–160Google Scholar
  99. 99.
    Weber W, Helms V, McCammon JA, Langhoff PW (1999) Shedding light on the dark and weakly fluorescent states of green fluorescent proteins. Proc Natl Acad Sci USA 96:6177–6182Google Scholar
  100. 100.
    Blum C, Subramaniam V (2009) Single-molecule spectroscopy of fluorescent proteins. Anal Bioanal Chem 393:527–541Google Scholar
  101. 101.
    Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388:355–358Google Scholar
  102. 102.
    Scharnagl C, Raupp-Kossmann RA (2004) Solution pK(a) values of the green fluorescent protein chromophore from hybrid quantum-classical calculations. J Phys Chem B 108:477–489Google Scholar
  103. 103.
    Petersen J, Wilmann PG, Beddoe T, Oakley AJ, Devenish RJ, Prescott M, Rossjohn J (2003) The 2.0-A crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J Biol Chem 278:44626–44631Google Scholar
  104. 104.
    Wiedenmann J, Schenk A, Rocker C, Girod A, Spindler KD, Nienhaus GU (2002) A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc Natl Acad Sci USA 99:11646–11651Google Scholar
  105. 105.
    Bravaya KB, Bochenkova AV, Granovsky AA, Savitsky AP, Nemukhin AV (2008) Modeling photoabsorption of the asFP595 chromophore. J Phys Chem A 112:8804–8810Google Scholar
  106. 106.
    Andresen M, Stiel AC, Folling J, Wenzel D, Schonle A, Egner A, Eggeling C, Hell SW, Jakobs S (2008) Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26:1035–1040Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.Division of Molecular BiosciencesImperial College LondonLondonUK

Personalised recommendations