One-Photon and Two-Photon Excitation of Fluorescent Proteins

  • R. NifosìEmail author
  • V. Tozzini
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)


Fluorescent proteins (FPs) offer a wide palette of colors for imaging applications. One purpose of this chapter is to review the variety of FP spectral properties, with a focus on their structural basis. Fluorescence in FPs originates from the autocatalytically formed chromophore. Several studies exist on synthetic chromophore analogs in gas phase and in solution. Together with the X-ray structures of many FPs, these studies help to understand how excitation and emission energies are tuned by chromophore structure, protonation state, and interactions with the surrounding environment, either solvent molecules or amino acids residues. The increasing use of FPs in two-photon microscopy also prompted detailed investigations of their two-photon excitation properties. The comparison with one-photon excitation reveals nontrivial features, which are relevant both for their implications in understanding multiphoton properties of fluorophores and for application purposes.


Fluorescent proteins Chromophore structures Computational studies Isolated chromophores Multiphoton spectroscopy Structure-property relationship Spectral tuning 


  1. 1.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of Aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239Google 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.
    Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D (1994) Green fluorescent protein as a marker for gene expression. Science 263(5148):802–805Google Scholar
  4. 4.
    Inouye S, Tsuji FI (1994) Aequorea green fluorescent protein: expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Lett 341:277–280Google Scholar
  5. 5.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544Google Scholar
  6. 6.
    Matz M, Fradkov AF, Labas Y, Savitsky A, Zaraisky AG, Markelov AZ, Lukyanov SA (1999) Fluorescent proteins from non-bioluminescent Anthozoa species. Nat Biotechnol 17:969–973Google Scholar
  7. 7.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909Google Scholar
  8. 8.
    Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73(5):2782–2790Google Scholar
  9. 9.
    Pakhomov AA, Martynov VI (2008) GFP family: structural insights into spectral tuning. Chem Biol 15(8):755–764Google Scholar
  10. 10.
    Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormö 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
  11. 11.
    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
  12. 12.
    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
  13. 13.
    Wachter RM (2007) Chromogenic cross-link formation in green fluorescent protein. Acc Chem Res 40(2):120–127Google Scholar
  14. 14.
    Wachter RM, Watkins JL, Kim H (2010) Mechanistic diversity of red fluorescence acquisition by GFP-like proteins. Biochemistry 49(35):7417–7427Google Scholar
  15. 15.
    Kummer AD, Kompa C, Lossau H, Pollinger-Dammer F, Michel-Beyerle ME, Silva CM, Bylina EJ, Coleman WJ, Yang MM, Youvan DC (1998) Dramatic reduction in fluorescence quantum yield in mutants of green fluorescent protein due to fast internal conversion. Chem Phys 237:183–193Google Scholar
  16. 16.
    Ward WW, Bokman SH (1982) Reversible denaturation of Aequorea green fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21:4535–4540Google Scholar
  17. 17.
    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
  18. 18.
    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
  19. 19.
    Bizzarri R, Nifosì R, Abbruzzetti S, Rocchia W, Guidi S, Arosio D, Garau G, Campanini B, Grandi E, Ricci F, Viappiani C, Beltram F (2007) Green fluorescent protein ground states: the influence of a second protonation site near the chromophore. Biochemistry 46(18):5494–5504Google Scholar
  20. 20.
    Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664Google Scholar
  21. 21.
    Ai H-W, Shaner NC, Cheng Z, Tsien RY, Campbell RE (2007) Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46(20):5904–5910Google Scholar
  22. 22.
    Mena MA, Treynor TP, Mayo SL, Daugherty PS (2006) Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nat Biotechnol 24(12):1569–1571Google Scholar
  23. 23.
    Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for fret. Nat Biotechnol 22(4):445–449Google Scholar
  24. 24.
    Ai H-W, Hazelwood KL, Davidson MW, Campbell RE (2008) Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat Methods 5(5):401–403Google Scholar
  25. 25.
    Zapata-Hommer O, Griesbeck O (2003) Efficiently folding and circularly permuted variants of the sapphire mutant of GFP. BMC Biotechnol 3:5Google Scholar
  26. 26.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306(5700):1370–1373Google Scholar
  27. 27.
    Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ (1998) Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6:1267–1277Google Scholar
  28. 28.
    Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90Google Scholar
  29. 29.
    Nguyen AW, Daugherty PS (2005) Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23:355–360Google Scholar
  30. 30.
    Shagin DA, Barsova EV, Yanushevich YG, Fradkov AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers A, Nunez JM, Widder EA, Lukyanov SA, Matz MV (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol 21(5):841–850Google Scholar
  31. 31.
    Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A (2004) Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J 381:307–312Google Scholar
  32. 32.
    Shaner N, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein. Nat Biotechnol 22:1567–1572Google Scholar
  33. 33.
    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(4):461–465Google Scholar
  34. 34.
    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(26):9156–9159Google Scholar
  35. 35.
    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
  36. 36.
    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(34):25879–25882Google Scholar
  37. 37.
    Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882Google Scholar
  38. 38.
    Wiedenmann J, Schenk A, Rcker C, Girod A, Spindler K-D, 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(18):11646–11651Google Scholar
  39. 39.
    Prescott M, Ling M, Beddoe T, Oakley AJ, Dove S, Hoegh-Guldberg O, Devenish RJ, Rossjohn J (2003) The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure 22:275–284Google Scholar
  40. 40.
    Wang L, Jackson WC, Steinbach PA, Tsien RY (2004) Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci USA 101(48):16745–16749Google Scholar
  41. 41.
    Lin MZ, McKeown MR, Ng H-L, Aguilera TA, Shaner NC, Campbell RE, Adams SR, Gross LA, Ma W, Alber T, Tsien RY (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16(11):1169–1179Google Scholar
  42. 42.
    Chan MCY, Karasawa S, Mizuno H, Bosanac I, Ho D, Priv GG, Miyawaki A, Ikura M (2006) Structural characterization of a blue chromoprotein and its yellow mutant from the sea anemone Cnidopus japonicus. J Biol Chem 281(49):37813–37819Google Scholar
  43. 43.
    Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY (2000) The structure of the chromophore within dsred, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97:11990–11995Google Scholar
  44. 44.
    Petersen J, Wilmann PG, Beddoe T, Oakley AJ, De-venish RJ, Prescott M, Rossjohn J (2003) The 2.0-angstrom crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J Biol Chem 287:44626–44631Google Scholar
  45. 45.
    Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45(32):9639–9647Google Scholar
  46. 46.
    Kikuchi A, Fukumura E, Karasawa S, Mizuno H, Miyawaki A, Shiro Y (2008) Structural characterization of a thiazoline-containing chromophore in an orange fluorescent protein, monomeric Kusabira Orange. Biochemistry 47(44):11573–11580Google Scholar
  47. 47.
    Remington SJ, Wachter RM, Yarbrough DK, Branchaud B, Anderson DC, Kallio K, Lukyanov KA (2005) zFP538, a yellow-fluorescent protein from Zoanthus, contains a novel three-ring chromophore. Biochemistry 44(1):202–212Google Scholar
  48. 48.
    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 Å resolution. Biochemistry 44(15):5774–5787Google Scholar
  49. 49.
    Tretyakova YA, Pakhomov AA, Martynov VI (2007) Chromophore structure of the kindling fluorescent protein asFP595 from Anemonia sulcata. J Am Chem Soc 129:7748–7749Google Scholar
  50. 50.
    Yampolsky IV, Remington SJ, Martynov VI, Potapov VK, Lukyanov S, Lukyanov KA (2005) Synthesis and properties of the chromophore of the asFP595 chromoprotein from Anemonia sulcata. Biochemistry 44(15):5788–5793Google Scholar
  51. 51.
    Schäfer LV, Groenhof G, Klingen AR, Ullmann GM, Boggio-Pasqua M, Robb MA, Grubmüller H (2007) Photoswitching of the fluorescent protein asFP595: mechanism, proton pathways, and absorption spectra. Angew Chem 119:536–542Google Scholar
  52. 52.
    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
  53. 53.
    He X, Bell AF, Tonge PJ (2002) Isotopic labeling and normal-mode analysis of a model green fluorescent protein chromophore. J Phys Chem B 106:6056–6066Google Scholar
  54. 54.
    Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, Kubota M, Ohashi M, Tsuji FI (1996) Chemical nature of the light emitter of the Aequorea green fluorescent protein. Proc Nat Acad Sci USA 93:13617–13622Google Scholar
  55. 55.
    Voliani V, Bizzarri R, Nifosì R, Abbruzzetti S, Grandi E, Viappiani C, Beltram F (2008) Cis–trans photoisomerization of fluorescent-protein chromophores. J Phys Chem B 112(34):10714–10722Google Scholar
  56. 56.
    Nielsen SB, Lapierre A, Andersen JU, Pedersen UV, Tomita S, Andersen LH (2001) Absorption spectrum of the green fluorescent protein chromophore anion in vacuo. Phys Rev Lett 87:228102Google Scholar
  57. 57.
    Dong J, Solntsev KM, Tolbert LM (2006) Solvatochromism of the green fluorescence protein chromophore and its derivatives. J Am Chem Soc 128(37):12038–12039Google Scholar
  58. 58.
    Yampolsky IV, Balashova TA, Lukyanov KA (2009) Synthesis and spectral and chemical properties of the yellow fluorescent protein zFP538 chromophore. Biochemistry 48:8077–8082Google Scholar
  59. 59.
    Boy S, Krogh H, Nielsen IB, Nielsen SB, Pedersen SU, Pedersen UV, Andersen LH, Bell AF, He X, Tonge PJ (2003) Vibrationally resolved photoabsorption spectroscopy of red fluorescent protein chromophore anions. Phys Rev Lett 90(11):118103Google Scholar
  60. 60.
    Yampolsky IV, Kislukhin AA, Amatov TT, Shcherbo D, Potapov VK, Lukyanov S, Lukyanov KA (2008) Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs. Bioorg Chem 36(2):96–104Google Scholar
  61. 61.
    Wachter RM, King BA, Heim R, Kallio K, Tsien RY, Boxer SG, Remington SJ (1997) Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein. Biochemistry 36:9759–9765Google Scholar
  62. 62.
    Forbes MW, Jockusch RA (2009) Deactivation pathways of an isolated green fluorescent protein model chromophore studied by electronic action spectroscopy. J Am Chem Soc 131(47):17038–17039Google Scholar
  63. 63.
    Boyé S, Nielsen SB, Krogh H, Nielsen IB, Pedersen UV, Bell AF, He X, Tonge PJ, Andersen LH (2003) Gas-phase absorption properties of DsRed model chromophores. Phys Chem Chem Phys 5(14):3021–3026Google Scholar
  64. 64.
    Andersen L, Lapierre A, Nielsen S, Nielsen I, Pedersen S, Pedersen U, Tomita S (2002) Chromophores of the green fluorescent protein studied in the gas phase. Eur Phys J D 20:597–600Google Scholar
  65. 65.
    Lammich L, Petersen MA, Nielsen MB, Andersen LH (2007) The gas-phase absorption spectrum of a neutral GFP model chromophore. Biophys J 92(1):201–207Google Scholar
  66. 66.
    Filippi C, Zaccheddu M, Buda F (2009) Absorption spectrum of the green fluorescent protein chromophore: a difficult case for ab initio methods? J Chem Theory Comput 5(8):2074–2087Google Scholar
  67. 67.
    Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J Phys Chem B 113(4):855–859Google Scholar
  68. 68.
    Mandal D, Tahara T, Meech SR (2004) Excited-state dynamics in the green fluorescent protein chromophore. J Phys Chem B 108(3):1102–1108Google Scholar
  69. 69.
    Voityuk AA, Michel-Beyerle M-E, Rosch N (1997) Protonation effects on the chromophore of green fluorescent protein. quantum chemical study of the absorption spectrum. Chem Phys Lett 272:162–167Google Scholar
  70. 70.
    Martin M, Negri F, Olivucci M (2004) Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution. J Am Chem Soc 126(17):5452–5464Google Scholar
  71. 71.
    Nifosì R, Amat P, Tozzini V (2007) Variation of spectral, structural and vibrational properties within the intrinsically fluorescent proteins family: a density functional study. J Comput Chem 28:2366–2377Google Scholar
  72. 72.
    Yan W, Zhang L, Xie D, Zeng J (2007) Electronic excitations of green fluorescent proteins: modeling solvatochromatic shifts of red fluorescent protein chromophore model compound in aqueous solutions. J Phys Chem B 111(50):14055–14063Google Scholar
  73. 73.
    Olsen S, Smith SC (2008) Bond selection in the photoisomerization reaction of anionic green fluorescent protein and kindling fluorescent protein chromophore models. J Am Chem Soc 130(27):8677–8689Google Scholar
  74. 74.
    Epifanovsky E, Polyakov I, Grigorenko B, Nemukhin A, Krylov AI (2009) Quantum chemical benchmark studies of the electronic properties of the green fluorescent protein chromophore. 1. Electronically excited and ionized states of the anionic chromophore in the gas phase. J Chem Theory Comput 5(7):1895–1906Google Scholar
  75. 75.
    Ma Y, Rohlfing M, Molteni C (2010) Modeling the excited states of biological chromophores within many-body green’s function theory. J Chem Theory Comput 6(1):257–265Google Scholar
  76. 76.
    Kowalski K, Krishnamoorthy S, Villa O, Hammond JR, Govind N (2010) Active-space completely-renormalized equation-of-motion coupled-cluster formalism: excited-state studies of green fluorescent protein, free-base porphyrin, and oligoporphyrin dimer. J Chem Phys 132(15):154103Google Scholar
  77. 77.
    Xie D, Zeng J (2005) Electronic excitations of green fluorescent proteins: protonation states of chromophore model compound in solutions. J Comput Chem 26(14):1487–1496Google Scholar
  78. 78.
    Wan S, Liu S, Zhao G, Chen M, Han K, Sun M (2007) Photoabsorption of green and red fluorescent protein chromophore anions in vacuo. Biophys Chem 129(2–3):218–223Google Scholar
  79. 79.
    Olsen S, Smith SC (2007) Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states. J Am Chem Soc 129(7):2054–2065Google Scholar
  80. 80.
    Sanchez-Garcia E, Doerr M, Thiel W (2010) QM/MM study of the absorption spectra of DsRed.m1 chromophores. J Comput Chem 31(8):1603–1612Google Scholar
  81. 81.
    Toniolo A, Granucci G, Martinez TJ (2003) Conical intersections in solution: a QM/MM study using floating occupation semiempirical configuration interaction wave functions. J Phys Chem A 107:3822Google Scholar
  82. 82.
    Drobizhev M, Tillo S, Makarov NS, Hughes TE, Rebane A (2009) Color hues in red fluorescent proteins are due to internal quadratic stark effect. J Phys Chem B 113(39):12860–12864Google Scholar
  83. 83.
    Hasegawa J-Y, Ise T, Fujimoto KJ, Kikuchi A, Fukumura E, Miyawaki A, Shiro Y (2010) Excited states of fluorescent proteins, mKO and DsRed: chromophore–protein electrostatic interaction behind the color variations. J Phys Chem B 114(8):2971–2979Google Scholar
  84. 84.
    He X, Bell AF, Tonge PJ (2002) Synthesis and spectroscopic studies of model red fluorescent protein chromophores. Org Lett 4(9):1523–1526Google Scholar
  85. 85.
    Laino T, Nifosì R, Tozzini V (2004) Relationship between structure and optical properties in green fluorescent proteins: an ab initio study of the active site. Chem Phys 298:17–28Google Scholar
  86. 86.
    Malo GD, Wang M, Wu D, Stelling AL, Tonge PJ, Wachter RM (2008) Crystal structure and Raman studies of dsFP483, a cyan fluorescent protein from Discosoma striata. J Mol Biol 378(4):871–886Google Scholar
  87. 87.
    Henderson JN, Remington SJ (2005) Crystal structures and mutational analysis of amFP486, a cyan fluorescent protein from Anemonia majano. Proc Natl Acad Sci USA 102(36):12712–12717Google Scholar
  88. 88.
    Henderson JN, Ai H-W, Campbell RE, Remington SJ (2007) Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc Natl Acad Sci USA 104(16):6672–6677Google Scholar
  89. 89.
    Ai H-W, Henderson JN, Remington SJ, Campbell RE (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J 400(3):531–540Google Scholar
  90. 90.
    Wood TI, Barondeau DP, Hitomi C, Kassmann CJ, Tainer JA, Getzoff ED (2005) Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry 44(49):16211–16220Google Scholar
  91. 91.
    Ai H-W, Campbell RE (2008) Teal fluorescent proteins: characterization of a reversibly photoswitchable variant. In: Proceedings of the SPIE – The International Society for Optical Engineering, pp 68680D1–7Google Scholar
  92. 92.
    Nakano H, Okumura R, Goto C, Yamane T (2002) In vitro combinatorial mutagenesis of the 65thand 222nd positions of the green fluorescent protein of Aequorea victoria. Biotechnol Bioprocess Eng 7:311–315Google Scholar
  93. 93.
    Delagrave S, Hawtin RE, Silva CM, Yang MM, Youvan DC (1995) Red-shifted excitation mutants of the green fluorescent protein. Biotechnology (N Y) 13(2):151–154Google Scholar
  94. 94.
    Labas Y, Gurskaya NG, Yanushevich YG, Fradkov AF, Lukyanov KA, Lukyanov SA, Matz MV (2002) Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci USA 99:4256–4261Google Scholar
  95. 95.
    Nienhaus K, Renzi F, Vallone B, Wiedenmann J, Nienhaus GU (2006) Exploring chromophore-protein interactions in fluorescent protein cmFP512 from Cerianthus membranaceus: X-ray structure analysis and optical spectroscopy. Biochemistry 45(43):12942–12953Google Scholar
  96. 96.
    Suto K, Masuda H, Takenaka Y, Tsuji FI, Mizuno H (2009) Structural basis for red-shifted emission of a GFP-like protein from the marine copepod Chiridius poppei. Genes Cells 14(6):727–737Google Scholar
  97. 97.
    Kummer AD, Wiehler J, Rehaber H, Kompa C, Steipe B, Michel-Beyerle ME (2000) Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP). J Phys Chem B 104:4791–4798Google Scholar
  98. 98.
    Nifosì R, Ferrari A, Arcangeli C, Tozzini V, Pellegrini V, Beltram F (2003) Photoreversible dark state in a tristable green fluorescent protein variant. J Phys Chem B 107:1679–1684Google Scholar
  99. 99.
    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(1):35–42Google Scholar
  100. 100.
    Shinobu A, Palm GJ, Schierbeek AJ, Agmon N (2010) Visualizing proton antenna in a high-resolution green fluorescent protein structure. J Am Chem Soc 132:11093–11102Google Scholar
  101. 101.
    Habuchi S, Dedecker P, Ichi Hotta J, Flors C, Ando R, Mizuno H, Miyawaki A, Hofkens J (2006) Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching. Photochem Photobiol Sci 5(6):567–576Google Scholar
  102. 102.
    Shu X, Kallio K, Shi X, Abbyad P, Kanchanawong P, Childs W, Boxer SG, Remington SJ (2007) Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 1. Mutagenesis and structural studies. Biochemistry 46(43):12005–12013Google Scholar
  103. 103.
    Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97:11984–11989Google Scholar
  104. 104.
    Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM (2009) Far-red fluorescent tags for protein imaging in living tissues. Biochem J 418(3):567–574Google Scholar
  105. 105.
    Tubbs JL, Tainer JA, Getzoff ED (2005) Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans–cis isomerization and acylimine formation in chromophore maturation. Biochemistry 44(29):9833–9840Google Scholar
  106. 106.
    Shu X, Wang L, Colip L, Kallio K, Remington SJ (2009) Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein. Protein Sci 18(2):460–466Google Scholar
  107. 107.
    Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S, Chudakov DM (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4(9):741–746Google Scholar
  108. 108.
    Blab GA, Lommerse PHM, Cognet L, Harms GS, Schmidt T (2001) Two-photon excitation action cross-sections of the autofluorescent proteins. Chem Phys Lett 350:71–77Google Scholar
  109. 109.
    Heikal AA, Hess ST, Webb WW (2001) Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid–base specificity. Chem Phys 274:37–55Google Scholar
  110. 110.
    Marchant JS, Stutzmann GE, Leissring MA, LaFerla FM, Parker I (2001) Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling. Nat Biotechnol 19:645–649Google Scholar
  111. 111.
    Tillo SE, Hughes TE, Makarov NS, Rebane A, Drobizhev M (2010) A new approach to dual-color two-photon microscopy with fluorescent proteins. BMC Biotechnol 10:6Google Scholar
  112. 112.
    Drobizhev M, Makarov NS, Hughes T, Rebane A (2007) Resonance enhancement of two-photon absorption in fluorescent proteins. J Phys Chem B 111(50):14051–14054Google Scholar
  113. 113.
    Nifosì R, Luo Y (2007) Predictions of novel two-photon absorption bands in fluorescent proteins. J Phys Chem B 111(50):14043–14050Google Scholar
  114. 114.
    Hosoi H, Yamaguchi S, Mizuno H, Miyawaki A, Tahara T (2008) Hidden electronic excited state of enhanced green fluorescent protein. J Phys Chem B 112(10):2761–2763Google Scholar
  115. 115.
    Cubitt AB, Woollenweber LA, Heim R (1999) Understanding structure-function relationships in the Aequorea Victoria Green Fluorescent Protein Method Cell Biol 58:19–30Google Scholar
  116. 116.
    Morise H, Shimomura O, Johnson FH, Winant J (1974) Intermolecular energy transfer in the bioluminescent system of Aequorea Biochemistry. American Chemical Society 13:2656–2662Google Scholar
  117. 117.
    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
  118. 118.
    Yang TT, Sinai P, Green G, Kitts PA, Chen YT, Lybarger L, Chervenak R, Patterson GH, Piston DW, Kain SR (1998) Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J Biol Chem 273:8212–8216Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.NEST CNR-NANOPisaItaly

Personalised recommendations