Primary Photophysical Processes in Chromoproteins

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


In this chapter, the diverse range of photophysical phenomena exhibited by chromoproteins is reviewed. Experimental and theoretical studies of both the electronic spectra and the ultrafast radiationless decay of the chromophore of green fluorescent protein (GFP) in solution are described as a function of solvent, temperature and substituent. The relevance of these observations to photophysical phenomena observed in chromoproteins which undergo photoconversion is discussed. Next, the excited state proton transfer found in GFP is described. Its potential to probe the dynamics of proton-transfer reactions in proteins is illustrated. Finally, the photophysics underlying the phenomenon of photoswitching in chromoproteins is discussed.


Fluorescence Fluorescent protein GFP Isomerization Photoswitching Proton transfer Ultrafast 



I am grateful to EPSRC for financial support (EP/H025715), to my students and postdoctoral fellows for their work and insights over the years, and to my collaborators for their generous advice and assistance.


  1. 1.
    Shimomura O, Johnson FH (1969) Properties of bioluminescent protein aequorin. Biochemistry 8:3991CrossRefGoogle Scholar
  2. 2.
    Morise H, Shimomur O, Johnson FH, Winant J (1974) Intermolecular energy-transfer in bioluminescent system of Aequorea. Biochemistry 13:2656–2662CrossRefGoogle Scholar
  3. 3.
    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene-expression. Science 263:802–805CrossRefGoogle Scholar
  4. 4.
    Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455CrossRefGoogle Scholar
  5. 5.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909CrossRefGoogle Scholar
  6. 6.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefGoogle Scholar
  7. 7.
    Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA (2010) Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 90:1103–1163Google Scholar
  8. 8.
    Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, Miller DJ, Wiedenmann J, Salih A, Matz MV (2008) Diversity and evolution of coral fluorescent proteins. PLoS One 3Google Scholar
  9. 9.
    Pouwels LJ, Zhang LP, 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 47:10111–10122CrossRefGoogle Scholar
  10. 10.
    Wachter RM, Watkins JL, Kim H (2010) Mechanistic diversity of red fluorescence acquisition by GFP-like proteins. Biochemistry 49:7417–7427Google Scholar
  11. 11.
    Wachter RM (2007) Chromogenic cross-link formation in green fluorescent protein. Acc Chem Res 40:120–127CrossRefGoogle Scholar
  12. 12.
    Shimomura O (1979) Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett 104:220–222CrossRefGoogle Scholar
  13. 13.
    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 Natl Acad Sci USA 93:13617–13622CrossRefGoogle Scholar
  14. 14.
    Frommer WB, Davidson MW, Campbell RE (2009) Genetically encoded biosensors based on engineered fluorescent proteins. Chem Soc Rev 38:2833–2841CrossRefGoogle Scholar
  15. 15.
    Meech SR (2009) Excited state reactions in fluorescent proteins. Chem Soc Rev 38:2922–2934CrossRefGoogle Scholar
  16. 16.
    van Thor JJ (2009) Photoreactions and dynamics of the green fluorescent protein. Chem Soc Rev 38:2935–2950CrossRefGoogle 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 39:4423–4431CrossRefGoogle Scholar
  18. 18.
    Elsliger MA, Wachter RM, Hanson GT, Kallio K, Remington SJ (1999) Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry 38:5296–5301CrossRefGoogle Scholar
  19. 19.
    Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 95:6803–6808CrossRefGoogle Scholar
  20. 20.
    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 93:8362–8367CrossRefGoogle Scholar
  21. 21.
    Sample V, Newman RH, Zhang J (2009) The structure and function of fluorescent proteins. Chem Soc Rev 38:2852–2864CrossRefGoogle Scholar
  22. 22.
    Lukyanov KA, Fradkov AF, Gurskaya NG, Matz MV, Labas YA, Savitsky AP, Markelov ML, Zaraisky AG, Zhao XN, Fang Y, Tan WY, Lukyanov SA (2000) Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem 275:25879–25882CrossRefGoogle Scholar
  23. 23.
    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 323:149–160CrossRefGoogle Scholar
  24. 24.
    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 105:18343–18348CrossRefGoogle Scholar
  25. 25.
    Kredel S, Nienhaus K, Oswald F, Wolff M, Ivanchenko S, Cymer F, Jeromin A, Michels FJ, Spindler KD, Heilker R, Nienhaus GU, Wiedenmannl J (2008) Optimized and far-red-emitting variants of fluorescent protein eqFP611. Chem Biol 15:224–233CrossRefGoogle Scholar
  26. 26.
    Nienhaus K, Nar H, Heilker R, Wiedenmann J, Nienhaus GU (2008) Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611. J Am Chem Soc 130:12578CrossRefGoogle Scholar
  27. 27.
    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 99:12651–12656CrossRefGoogle Scholar
  28. 28.
    Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One 3Google Scholar
  29. 29.
    Mizuno H, Mal TK, Tong KI, Ando R, Furuta T, Ikura M, Miyawakil A (2003) Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell 12:1051–1058CrossRefGoogle Scholar
  30. 30.
    Vaziri A, Tang JY, Shroff H, Shank CV (2008) Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci USA 105:20221–20226CrossRefGoogle Scholar
  31. 31.
    Bates M, Huang B, Dempsey GT, Zhuang XW (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317:1749–1753CrossRefGoogle Scholar
  32. 32.
    Patterson G, Davidson M, Manley S, Lippincott-Schwartz J (2010) Annu Rev Phys Chem 61:345–367Google Scholar
  33. 33.
    Rulliere C (ed) (2003) Femtosecond laser pulses: principles and experiments. Springer, New YorkGoogle Scholar
  34. 34.
    Diels JC, Rudolphe W (2006) Ultrashort laser pulse phenomena. Academic, New YorkGoogle Scholar
  35. 35.
    Andrews DL, Allcock P (2002) Optical harmonics in molecular systems. Wiley-VCH, WeinheimCrossRefGoogle Scholar
  36. 36.
    Fleming GR (1986) Chemical applications of ultrafast spectroscopy. Oxford University Press, OxfordGoogle Scholar
  37. 37.
    Rhee H, Joo T (2005) Noncolinear phase matching in fluorescence upconversion. Opt Lett 30:96–98CrossRefGoogle Scholar
  38. 38.
    Heisler IA, Kondo M, Meech SR (2009) Reactive dynamics in confined liquids: ultrafast torsional dynamics of auramine O in nanoconfined water in aerosol OT reverse micelles. J Phys Chem B 113:1623–1631CrossRefGoogle Scholar
  39. 39.
    Vengris M, van der Horst MA, Zgrablic G, van Stokkum IHM, Haacke S, Chergui M, Hellingwerf KJ, van Grondelle R, Larsen DS (2004) Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments. Biophys J 87:1848–1857CrossRefGoogle Scholar
  40. 40.
    Arzhantsev S, Zachariasse KA, Maroncelli M (2006) Photophysics of trans-4-(dimethylamino)-4′-cyanostilbene and its use as a solvation probe. J Phys Chem A 110:3454–3470CrossRefGoogle Scholar
  41. 41.
    Kwok WM, Ma C, Phillips D, Matousek P, Parker AW, Towrie M (2000) Picosecond time-resolved study of 4-dimethylaminobenzonitrile in polar and nonpolar solvents. J Phys Chem A 104:4188–4197CrossRefGoogle Scholar
  42. 42.
    Schmidt B, Laimgruber S, Zinth W, Gilch P (2003) A broadband Kerr shutter for femtosecond fluorescence spectroscopy. Appl Phys B Lasers Opts 76:809–814CrossRefGoogle Scholar
  43. 43.
    Takeda J, Nakajima K, Kurita S, Tomimoto S, Saito S, Suemoto T (2000) Femtosecond optical Kerr gate fluorescence spectroscopy for ultrafast relaxation processes. J Lumin 87–89:927–929CrossRefGoogle Scholar
  44. 44.
    van Stokkum IHM, Gobets B, Gensch T, van Mourik F, Hellingwerf KJ, van Grondelle R, Kennis JTM (2006) (Sub)-picosecond spectral evolution of fluorescence in photoactive proteins studied with a synchroscan streak camera system. Photochem Photobiol 82:380–388CrossRefGoogle Scholar
  45. 45.
    Schmitt M, Dietzek B, Hermann G, Popp J (2007) Femtosecond time-resolved spectroscopy on biological photoreceptor chromophores. Laser Photon Rev 1:57–78CrossRefGoogle Scholar
  46. 46.
    Greetham G, Towrie M, Matousek P, Parker AW (2010) J Appl Spectrosc 64:1320CrossRefGoogle Scholar
  47. 47.
    Towrie M, Grills DC, Dyer J, Weinstein JA, Matousek P, Barton R, Bailey PD, Subramaniam N, Kwok WM, Ma CS, Phillips D, Parker AW, George MW (2003) Development of a broadband picosecond infrared spectrometer and its incorporation into an existing ultrafast time-resolved resonance Raman, UV/visible, and fluorescence spectroscopic apparatus. Appl Spectrosc 57:367–380CrossRefGoogle Scholar
  48. 48.
    van Stokkum IHM, Larsen DS, van Grondelle R (2004) Global and target analysis of time-resolved spectra. Biochim Biophys Acta Bioenergetics 1657:82–104CrossRefGoogle Scholar
  49. 49.
    Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721CrossRefGoogle Scholar
  50. 50.
    van Thor JJ, Pierik AJ, Nugteren-Roodzant I, Xie AH, Hellingwerf KJ (1998) Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy. Biochemistry 37:16915–16921CrossRefGoogle Scholar
  51. 51.
    Bell AF, Stoner-Ma D, Wachter RM, Tonge PJ (2003) Light-driven decarboxylation of wild-type green fluorescent protein. J Am Chem Soc 125:6919–6926CrossRefGoogle Scholar
  52. 52.
    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. Struct Folding Design 6:1267–1277CrossRefGoogle Scholar
  53. 53.
    Andersen LH, Lapierre A, Nielsen SB, Nielsen IB, Pedersen SU, Pedersen UV, Tomita S (2002) Chromophores of the green fluorescent protein studied in the gas phase. Eur Phys J D 20:597–600CrossRefGoogle Scholar
  54. 54.
    Lammich L, Petersen MA, Nielsen MB, Andersen LH (2007) The gas-phase absorption spectrum of a neutral GFP model chromophore. Biophys J 92:201–207CrossRefGoogle Scholar
  55. 55.
    Forbes MW, Jockusch RA (2009) Deactivation pathways of an isolated green fluorescent protein model chromophore studied by electronic action spectroscopy. J Am Chem Soc 131:17038CrossRefGoogle Scholar
  56. 56.
    Webber NM, Meech SR (2007) Electronic spectroscopy and solvatochromism in the chromophore of GFP and the Y66F mutant. Photochem Photobiol Sci 9:276–281Google Scholar
  57. 57.
    Dong J, Solntsev KM, Tolbert LM (2006) Solvatochromism of the green fluorescence protein chromophore and its derivatives. J Am Chem Soc 128:12038–12039CrossRefGoogle Scholar
  58. 58.
    Conyard J, Kondo M, Heisler IA, Baldridge A, Tolbert LM, Solntsev KM, Meech SR (2011) J Phys Chem B 115:1863–1873Google Scholar
  59. 59.
    Gepshtein R, Huppert D, Agmon N (2006) Deactivation mechanism of the green fluorescent chromophore. J Phys Chem B 110:4434–4442CrossRefGoogle Scholar
  60. 60.
    Vengris M, van Stokkum IHM, He X, Bell AF, Tonge PJ, van Grondelle R, Larsen DS (2004) Ultrafast excited and ground-state dynamics of the green fluorescent protein chromophore in solution. J Phys Chem A 108:4587–4598CrossRefGoogle Scholar
  61. 61.
    Litvinenko KL, Webber NM, Meech SR (2003) Internal conversion in the chromophore of the green fluorescent protein: temperature dependence and isoviscosity analysis. J Phys Chem A 107:2616–2623CrossRefGoogle Scholar
  62. 62.
    Mandal D, Tahara T, Meech SR (2004) Excited-state dynamics in the green fluorescent protein chromophore. J Phys Chem B 108:1102–1108CrossRefGoogle Scholar
  63. 63.
    Mandal D, Tahara T, Webber NM, Meech SR (2002) Ultrafast fluorescence of the chromophore of the green fluorescent protein in alcohol solutions. Chem Phys Lett 358:495–501CrossRefGoogle Scholar
  64. 64.
    Webber NM, Litvinenko KL, Meech SR (2001) Radiationless relaxation in a synthetic analogue of the green fluorescent protein chromophore. J Phys Chem B 105:8036–8039CrossRefGoogle Scholar
  65. 65.
    Kummer AD, Kompa C, Niwa H, Hirano T, Kojima S, Michel-Beyerle ME (2002) Viscosity-dependent fluorescence decay of the GFP chromophore in solution due to fast internal conversion. J Phys Chem B 106:7554–7559CrossRefGoogle Scholar
  66. 66.
    Sension RJ, Repinec ST, Szarka AZ, Hochstrasser RM (1993) Femtosecond laser studies of the cis-stilbene photoisomerization reactions. J Chem Phys 98:6291–6315CrossRefGoogle Scholar
  67. 67.
    Kim SK, Fleming GR (1988) Reorientation and isomerization of trans-stilbene in alkane solutions. J Phys Chem 92:2168–2172CrossRefGoogle Scholar
  68. 68.
    Nagele T, Hoche R, Zinth W, Wachtveitl J (1997) Femtosecond photoisomerization of cis-azobenzene. Chem Phys Lett 272:489–495CrossRefGoogle Scholar
  69. 69.
    Sundstrom V, Gillbro T (1982) Viscosity-dependent isomerization yields of some cyanine dyes – a picosecond laser spectroscopy study. J Phys Chem 86:1788–1794CrossRefGoogle Scholar
  70. 70.
    He X, Bell AF, Tonge PJ (2003) Ground state isomerization of a model green fluorescent protein chromophore. FEBS Lett 549:35–38CrossRefGoogle Scholar
  71. 71.
    Dong J, Abulwerdi F, Baldridge A, Kowalik J, Solntsev KM, Tolbert LM (2008) Isomerization in fluorescent protein chromophores involves addition/elimination. J Am Chem Soc 130:14096CrossRefGoogle Scholar
  72. 72.
    Voliani V, Bizzarri R, Nifosi R, Abbruzzetti S, Grandi E, Viappiani C, Beltram F (2008) Cis-trans photoisomerization of fluorescent-protein chromophores. J Phys Chem B 112:10714–10722CrossRefGoogle Scholar
  73. 73.
    Yang JS, Huang GJ, Liu YH, Peng SM (2008) Photoisomerization of the green fluorescence protein chromophore and the meta- and para-amino analogues. Chem Commun:1344–1346Google Scholar
  74. 74.
    Huang GJ, Yang JS (2010) The N-arylamino conjugation effect in the photochemistry of fluorescent protein chromophores and aminostilbenes. Chem Asian J 5:2075–2085Google Scholar
  75. 75.
    Wang DQ, Merz T, van Gunsteren WF (2010) The thermal isomerization of the GFP chromophore: a computational study. Phys Chem Chem Phys 12:11051–11061.Google Scholar
  76. 76.
    Wu LX, Burgess K (2008) Syntheses of highly fluorescent GFP-chromophore analogues. J Am Chem Soc 130:4089–4096CrossRefGoogle Scholar
  77. 77.
    Stavrov SS, Solntsev KM, Tolbert LM, Huppert D (2006) Probing the decay coordinate of the green fluorescent protein: arrest of cis-trans isomerization by the protein significantly narrows the fluorescence spectra. J Am Chem Soc 128:1540–1546CrossRefGoogle Scholar
  78. 78.
    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 96:6177–6182CrossRefGoogle Scholar
  79. 79.
    Altoe P, Bernardi F, Garavelli M, Orlandi G, Negri F (2005) Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: a quantum-chemical study. J Am Chem Soc 127:3952–3963CrossRefGoogle Scholar
  80. 80.
    Martin ME, 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 126:5452–5464CrossRefGoogle Scholar
  81. 81.
    Toniolo A, Olsen S, Manohar L, Martinez TJ (2004) Conical intersection dynamics in solution: the chromophore of green fluorescent protein. Farad Discuss 127:149–163CrossRefGoogle Scholar
  82. 82.
    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
  83. 83.
    Olsen S, Manohar L, Martinez TJ (2002) Features of interest on the S-0 and S-1 potential energy surfaces of a model green fluorescent protein chromophore. Biophys J 82:359A–359AGoogle Scholar
  84. 84.
    Olsen S, Smith SC (2007) Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states. J Am Chem Soc 129:2054–2065CrossRefGoogle Scholar
  85. 85.
    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 130:8677–8689CrossRefGoogle Scholar
  86. 86.
    Baffour-Awuah NYA, Zimmer M (2004) Hula-twisting in green fluorescent protein. Chem Phys 303:7–11CrossRefGoogle Scholar
  87. 87.
    Megley CM, Dickson LA, Maddalo SL, Chandler GJ, Zimmer M (2009) Photophysics and dihedral freedom of the chromophore in yellow, blue, and green fluorescent protein. J Phys Chem B 113:302–308CrossRefGoogle Scholar
  88. 88.
    Wilmann PG, Petersen J, Pettikiriarachchi A, Buckle AM, Smith SC, Olsen S, Perugini MA, Devenish RJ, Prescott M, Rossjohn J (2005) The 2.1 angstrom crystal structure of the far-red fluorescent protein HcRed: inherent conformational flexibility of the chromophore. J Mol Biol 349:223–237CrossRefGoogle 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 104:6672–6677CrossRefGoogle Scholar
  90. 90.
    Prescott M, Ling M, Beddoe T, Oakley AJ, Dove S, Hoegh-Guldberg O, Devenish RJ, Rossjohn J (2003) The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure 11:275–284CrossRefGoogle Scholar
  91. 91.
    Jaye AA, Stoner-Ma D, Matousek P, Towrie M, Tonge PJ, Meech SR (2006) Time-resolved emission spectra of green fluorescent protein. Photochem Photobiol 82:373–379Google Scholar
  92. 92.
    Arnaut LG, Formosinho SJ (1993) Excited-state proton-transfer reactions. 1. Fundamentals and intermolecular reactions. J Photochem Photobiol A Chem 75:1–20Google Scholar
  93. 93.
    Lossau H, Kummer A, Heinecke R, PollingerDammer F, Kompa C, Bieser G, Jonsson T, Silva CM, Yang MM, Youvan DC, MichelBeyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore-protein interactions. Chem Phys 213:1–16CrossRefGoogle Scholar
  94. 94.
    Kennis JTM, Larsen DS, van Stokkum NHM, Vengris M, van Thor JJ, van Grondelle R (2004) Uncovering the hidden ground state of green fluorescent protein. Proc Natl Acad Sci USA 101:17988–17993Google Scholar
  95. 95.
    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 94:2306–2311CrossRefGoogle Scholar
  96. 96.
    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 127:2864–2865CrossRefGoogle Scholar
  97. 97.
    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 110:22009–22018CrossRefGoogle Scholar
  98. 98.
    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 280:33652–33659CrossRefGoogle Scholar
  99. 99.
    van Thor JJ, Zanetti G, Ronayne KL, Towrie M (2005) Structural events in the photocycle of green fluorescent protein. J Phys Chem B 109:16099–16108CrossRefGoogle Scholar
  100. 100.
    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 95:1902–1912CrossRefGoogle Scholar
  101. 101.
    Frank RAW, Titman CM, Pratap JV, Luisi BF, Perham RN (2004) A molecular switch and proton wire synchronize the active sites in thiamine enzymes. Science 306:872–876CrossRefGoogle Scholar
  102. 102.
    Burykin A, Warshel A (2003) What really prevents proton transport through aquaporin? Charge self-energy versus proton wire proposals. Biophys J 85:3696–3706CrossRefGoogle Scholar
  103. 103.
    Cui Q, Karplus M (2003) Is a “proton wire” concerted or stepwise? A model study of proton transfer in carbonic anhydrase. J Phys Chem B 107:1071–1078CrossRefGoogle Scholar
  104. 104.
    Stoner-Ma D, Jaye AA, Ronayne KL, Nappa J, Tonge PJ, Meech SR (2008) Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): snipping the proton wire. Chem Phys 350:193–200CrossRefGoogle Scholar
  105. 105.
    Shi X, Abbyad P, Shu X, Kallio K, Kanchanawong P, Childs W, Remington SJ, Boxer SG (2007) Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 2. Unusual photophysical properties. Biochemistry 46:12014–12025CrossRefGoogle Scholar
  106. 106.
    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 46:12005–12013CrossRefGoogle Scholar
  107. 107.
    Leiderman P, Genosar L, Huppert D, Shu X, Remington SJ, Solntsev KM, Tolbert LM (2007) Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer. Biochemistry 46:12026–12036CrossRefGoogle Scholar
  108. 108.
    Stoner-Ma D, Jaye AA, Ronayne KL, Nappa J, Meech SR, Tonge PJ (2008) An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP. J Am Chem Soc 130:1227–1235CrossRefGoogle Scholar
  109. 109.
    Kondo M, Meech SR, Tonge PJ, Stoner-Ma D, Heisler IA (2010) Ultrafast dynamics of protein proton transfer on short hydrogen bond potential energy surfaces: S65T/H148D GFP. J Am Chem Soc 152:1452–1453CrossRefGoogle Scholar
  110. 110.
    Cleland WW, Kreevoy MM (1994) Low-barrier hydrogen-bonds and enzymatic catalysis. Science 264:1887–1890CrossRefGoogle Scholar
  111. 111.
    Cleland WW (2010) Adv Phys Org Chem 44:1–17Google Scholar
  112. 112.
    Lill MA, Helms V (2002) Proton shuttle in green fluorescent protein studied by dynamic simulations. Proc Natl Acad Sci USA 99:2778–2781CrossRefGoogle Scholar
  113. 113.
    Wang SF, Smith SC (2006) Leading coordinate analysis of reaction pathways in proton chain transfer: application to a two-proton transfer model for the green fluorescent protein. Chem Phys 326:204–209CrossRefGoogle Scholar
  114. 114.
    Wang SF, Smith SC (2007) Mechanistic aspects of proton chain transfer in the green fluorescent protein – Part II. A comparison of minimal quantum chemical models. Phys Chem Chem Phys 9:452–458CrossRefGoogle Scholar
  115. 115.
    Zhang H, Smith SC (2007) Model real-time quantum dynamical simulations of proton transfer in the green fluorescent protein (GFP). J Theor Comput Chem 6:789–802CrossRefGoogle Scholar
  116. 116.
    Zhang RB, Nguyen MT, Ceulemans A (2005) A concerted mechanism of proton transfer in green fluorescent protein. A theoretical study. Chem Phys Lett 404:250–256CrossRefGoogle Scholar
  117. 117.
    Vendrell O, Gelabert R, Moreno M, Lluch JM (2008) A potential energy function for heterogeneous proton-wires. Ground end photoactive states of the proton-wire in the green fluorescent protein. J Chem Theory Comput 4:1138–1150CrossRefGoogle Scholar
  118. 118.
    Vendrell O, Gelabert R, Moreno M, Lluch JM (2008) Exploring the effects of intramolecular vibrational energy redistribution on the operation of the proton wire in green fluorescent protein. J Phys Chem B 112:13443–13452CrossRefGoogle Scholar
  119. 119.
    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 112:5500–5511CrossRefGoogle Scholar
  120. 120.
    Fang C, Frontiera RR, Tran R, Mathies RA (2009) Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy. Nature 462:200–204CrossRefGoogle Scholar
  121. 121.
    Irie M (2000) Chem Rev 100(5):1685–1716Google Scholar
  122. 122.
    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373CrossRefGoogle Scholar
  123. 123.
    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 24:461–465CrossRefGoogle Scholar
  124. 124.
    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 101:15905–15910CrossRefGoogle Scholar
  125. 125.
    Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120:4247–4260CrossRefGoogle Scholar
  126. 126.
    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 372:918–926CrossRefGoogle Scholar
  127. 127.
    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 131:16814–16823CrossRefGoogle Scholar
  128. 128.
    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 6:233–238CrossRefGoogle Scholar
  129. 129.
    Lukacs A, Kondo M, Heisler IA, Miyawaki A, Tsutsui H, Towrie M, Greetham G, Tonge PJ, Stoner-Ma D, Meech SR (2010) In: Ultrafast phenomena XVII. Jonas D, Riedle E, Schoenlein R, Chergui M, Taylor A (eds) OUP: 511–513Google Scholar
  130. 130.
    Dedecker P, Hotta J, Ando R, Miyawaki A, Engelborghs Y, Hofkens J (2006) Fast and reversible photoswitching of the fluorescent protein Dronpa as evidenced by fluorescence correlation spectroscopy. Biophys J 91:L45–L47CrossRefGoogle Scholar
  131. 131.
    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 102:9511–9516CrossRefGoogle Scholar
  132. 132.
    Habuchi S, Dedecker P, Hotta JI, 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 5:567–576CrossRefGoogle Scholar
  133. 133.
    Subach FV, Zhang LJ, Gadella TWJ, Gurskaya NG, Lukyanov KA, Verkhusha VV (2010) Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chem Biol 17:745–755Google Scholar
  134. 134.
    Stiel AC, Andresen M, Bock H, Hilbert M, Schilde J, Schonle A, Eggeling C, Egner A, Hell SW, Jakobs S (2008) Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys J 95:2989–2997CrossRefGoogle Scholar
  135. 135.
    Ando R, Flors C, Mizuno H, Hofkens J, Miyawaki A (2007) Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants. Biophys J 92:L97–L99CrossRefGoogle Scholar
  136. 136.
    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 105:9227–9232CrossRefGoogle Scholar
  137. 137.
    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 129:4870CrossRefGoogle Scholar
  138. 138.
    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 104:13005–13009CrossRefGoogle Scholar
  139. 139.
    Stiel AC, Trowitzsch S, Weber G, Andresen M, Eggeling C, Hell SW, Jakobs S, Wahl MC (2007) 1.8 Angstrom bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem J 402:35–42CrossRefGoogle Scholar
  140. 140.
    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
  141. 141.
    Bulina ME, Chudakov DM, Britanova OV, Yanushevich YG, Staroverov DB, Chepurnykh TV, Merzlyak EM, Shkrob MA, Lukyanov S, Lukyanov KA (2006) A genetically encoded photosensitizer. Nat Biotechnol 24:95–99CrossRefGoogle Scholar
  142. 142.
    Kent KP, Childs W, Boxer SG (2008) Deconstructing green fluorescent protein. J Am Chem Soc 130:9664CrossRefGoogle Scholar
  143. 143.
    Kent KP, Oltrogge LM, Boxer SG (2009) Synthetic control of green fluorescent protein. J Am Chem Soc 131:15988CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.School of ChemistryUniversity of East AngliaNorwichUK

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