Advertisement

Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level

  • Christian BlumEmail author
  • Vinod Subramaniam
Chapter
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)

Abstract

The photophysics of visible fluorescent proteins (VFPs) remains a topic of intense research, driven by the widespread use of these proteins as reporters and sensors in living cells. The photophysical complexity of these markers originates from the multistep chemical reaction that forms the chromophore, and from the embedding of the chromophore within the protein nanoenvironment. To accurately interpret the biological and biochemical processes illuminated by the VFPs, it is essential to understand the details of their photophysics. Certain aspects of VFP photophysics can only be observed and understood at the single molecule level, which removes the averaging effect inherent to ensemble studies. Here, we review how spectrally resolved single molecule emission detection at room temperature has helped to understand the complex photophysics of VFPs. We focus on the detection of spectrally distinct subensembles, the spontaneous or light-induced transition between these subensembles, and on subtle spectral variations induced by changes in the local nanoenvironment of VFP chromophores.

Keywords

Fluorescence spectra Photophysics Photochromism Single molecule spectroscopy Spectral diffusion Spectral forms 

References

  1. 1.
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Science 313(5793):1642–1645CrossRefGoogle Scholar
  2. 2.
    Donnert G, Keller J, Wurm CA, Rizzoli SO, Westphal V, Schonle A, Jahn R, Jakobs S, Eggeling C, Hell SW (2007) Biophys J 92(8):L67–L69CrossRefGoogle Scholar
  3. 3.
    Hell SW (2007) Science 316(5828):1153–1158CrossRefGoogle Scholar
  4. 4.
    Hess ST, Girirajan TP, Mason MD (2006) Biophys J 91(11):4258–4272CrossRefGoogle Scholar
  5. 5.
    Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, Kner P, Burke B, Cardoso MC, Agard DA, Gustafsson MG, Leonhardt H, Sedat JW (2008) Science 320(5881):1332–1336CrossRefGoogle Scholar
  6. 6.
    Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Nat Methods 5(5):417–423CrossRefGoogle Scholar
  7. 7.
    Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW (2006) Nat Methods 3(9):721–723CrossRefGoogle Scholar
  8. 8.
    Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Nat Biotechnol 17(10):969–973CrossRefGoogle Scholar
  9. 9.
    Gurskaya NG, Fradkov AF, Terskikh A, Matz MV, Labas YA, Martynov VI, Yanushevich YG, Lukyanov KA, Lukyanov SA (2001) FEBS Lett 507(1):16–20CrossRefGoogle Scholar
  10. 10.
    Wiedenmann J, Schenk A, Rocker C, Girod A, Spindler KD, Nienhaus GU (2002) Proc Natl Acad Sci USA 99(18):11646–11651CrossRefGoogle Scholar
  11. 11.
    Karasawa S, Araki T, Yamamoto-Hino M, Miyawaki A (2003) J Biol Chem 278(36):34167–34171CrossRefGoogle Scholar
  12. 12.
    Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, Miyawaki A (2006) Nat Biotechnol 24(5):577–581CrossRefGoogle Scholar
  13. 13.
    Chan MC, Karasawa S, Mizuno H, Bosanac I, Ho D, Prive GG, Miyawaki A, Ikura M (2006) J Biol Chem 281(49):37813–37819CrossRefGoogle Scholar
  14. 14.
    Beddoe T, Ling M, Dove S, Hoegh-Guldberg O, Devenish RJ, Prescott M, Rossjohn J (2003) Acta Crystallogr D Biol Crystallogr 59(Pt 3):597–599CrossRefGoogle Scholar
  15. 15.
    Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME, Shcheglov AS, Fradkov AF, Gaintzeva A, Lukyanov KA, Lukyanov S, Gadella TW, Chudakov DM (2007) Nat Methods 4(7):555–557CrossRefGoogle Scholar
  16. 16.
    Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF, Ermakova GV, Solovieva EA, Lukyanov KA, Bogdanova EA, Zaraisky AG, Lukyanov S, Chudakov DM (2007) Nat Methods 4(9):741–746CrossRefGoogle Scholar
  17. 17.
    Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) Proc Natl Acad Sci USA 99(12):7877–7882CrossRefGoogle Scholar
  18. 18.
    Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao XN, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P (2000) Science 290(5496):1585–1588CrossRefGoogle Scholar
  19. 19.
    Bevis BJ, Glick BS (2002) Nat Biotechnol 20(1):83–87CrossRefGoogle Scholar
  20. 20.
    Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Nat Biotechnol 22(12):1567–1572CrossRefGoogle Scholar
  21. 21.
    Lippincott-Schwartz J, Patterson GH (2008) Methods Cell Biol 85:45–61CrossRefGoogle Scholar
  22. 22.
    Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Nat Cell Biol 5(Suppl):S7–S14Google Scholar
  23. 23.
    Patterson GH, Lippincott-Schwartz J (2002) Science 297(5588):1873–1877CrossRefGoogle Scholar
  24. 24.
    Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, Zorov DB, Lukyanov S, Lukyanov KA (2003) Nat Biotechnol 21(2):191–194CrossRefGoogle Scholar
  25. 25.
    Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA (2004) Nat Biotechnol 22(11):1435–1439CrossRefGoogle Scholar
  26. 26.
    Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005) Nat Rev Mol Cell Biol 6(11):885–891CrossRefGoogle Scholar
  27. 27.
    Chudakov DM, Chepurnykh TV, Belousov VV, Lukyanov S, Lukyanov KA (2006) Traffic 7(10):1304–1310CrossRefGoogle Scholar
  28. 28.
    Flors C, Hotta J, Uji-i H, Dedecker P, Ando R, Mizuno H, Miyawaki A, Hofkens J (2007) J Am Chem Soc 129(45):13970–13977CrossRefGoogle Scholar
  29. 29.
    Sinnecker D, Voigt P, Hellwig N, Schaefer M (2005) Biochemistry 44(18):7085–7094CrossRefGoogle Scholar
  30. 30.
    Henderson JN, Ai HW, Campbell RE, Remington SJ (2007) Proc Natl Acad Sci USA 104(16):6672–6677CrossRefGoogle Scholar
  31. 31.
    Bulina ME, Chudakov DM, Britanova OV, Yanushevich YG, Staroverov DB, Chepurnykh TV, Merzlyak EM, Shkrob MA, Lukyanov S, Lukyanov KA (2006) Nat Biotechnol 24(1):95–99CrossRefGoogle Scholar
  32. 32.
    Bulina ME, Lukyanov KA, Britanova OV, Onichtchouk D, Lukyanov S, Chudakov DM (2006) Nat Protoc 1(2):947–953CrossRefGoogle Scholar
  33. 33.
    Weiss S (1999) Science 283(5408):1676–1683CrossRefGoogle Scholar
  34. 34.
    Moerner WE, Fromm DP (2003) Rev Sci Instrum 74(8):3597–3619CrossRefGoogle Scholar
  35. 35.
    Garcia-Parajo MF, Veerman JA, Bouwhuis R, Vallee R, van Hulst NF (2001) Chemphyschem 2(6):347–360CrossRefGoogle Scholar
  36. 36.
    Blum C, Meixner AJ, Subramaniam V (2004) Biophys J 87(6):4172–4179CrossRefGoogle Scholar
  37. 37.
    Yang F, Moss LG, Phillips GN (1996) Nat Biotechnol 14(10):1246–1251CrossRefGoogle Scholar
  38. 38.
    Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Science 273(5280):1392–1395CrossRefGoogle Scholar
  39. 39.
    Verkhusha VV, Chudakov DM, Gurskaya NG, Lukyanov S, Lukyanov KA (2004) Chem Biol 11(6):845–854CrossRefGoogle Scholar
  40. 40.
    Strack RL, Strongin DE, Mets L, Glick BS, Keenan RJ (2010) J Am Chem Soc 132(24):8496–8505CrossRefGoogle Scholar
  41. 41.
    Remington SJ, Wachter RM, Yarbrough DK, Branchaud B, Anderson DC, Kallio K, Lukyanov KA (2005) Biochemistry 44(1):202–212CrossRefGoogle Scholar
  42. 42.
    Shu X, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ (2006) Biochemistry 45(32):9639–9647CrossRefGoogle Scholar
  43. 43.
    Tubbs JL, Tainer JA, Getzoff ED (2005) Biochemistry 44(29):9833–9840CrossRefGoogle Scholar
  44. 44.
    Terskikh AV, Fradkov AF, Zaraisky AG, Kajava AV, Angres B (2002) J Biol Chem 277(10):7633–7636CrossRefGoogle Scholar
  45. 45.
    van Thor JJ, Gensch T, Hellingwerf KJ, Johnson LN (2002) Nat Struct Biol 9(1):37–41CrossRefGoogle Scholar
  46. 46.
    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) J Am Chem Soc 127(25):8977–8984CrossRefGoogle Scholar
  47. 47.
    Bonsma S, Gallus J, Konz F, Purchase R, Volker S (2004) J Lumin 107(1–4):203–212CrossRefGoogle Scholar
  48. 48.
    Creemers TMH, Lock AJ, Subramaniam V, Jovin TM, Volker S (2000) Proc Natl Acad Sci USA 97(7):2974–2978CrossRefGoogle Scholar
  49. 49.
    Tsien RY (1998) Annu Rev Biochem 67:509–544CrossRefGoogle Scholar
  50. 50.
    Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ (1998) Structure Fold Des 6(10):1267–1277CrossRefGoogle Scholar
  51. 51.
    Blum C, Meixner AJ, Subramaniam V (2006) J Am Chem Soc 128(26):8664–8670CrossRefGoogle Scholar
  52. 52.
    Jung G, Wiehler J, Zumbusch A (2005) Biophys J 88(3):1932–1947CrossRefGoogle Scholar
  53. 53.
    Jung G, Ma YZ, Prall BS, Fleming GR (2005) Chemphyschem 6(8):1628–1632CrossRefGoogle Scholar
  54. 54.
    Wiedenmann J, Ivanchenko S, Oswald F, Nienhaus GU (2004) Mar Biotechnol (NY) 6(3):270–277CrossRefGoogle Scholar
  55. 55.
    Roy R, Hohng S, Ha T (2008) Nat Meth 5(6):507–516CrossRefGoogle Scholar
  56. 56.
    Schneckenburger H (2005) Curr Opin Biotechnol 16(1):13–18CrossRefGoogle Scholar
  57. 57.
    Nie SM, Zare RN (1997) Annu Rev Biophys Biomol Struct 26:567–596CrossRefGoogle Scholar
  58. 58.
    Dickson RM, Cubitt AB, Tsien RY, Moerner WE (1997) Nature 388(6640):355–358CrossRefGoogle Scholar
  59. 59.
    Peterman EJG, Brasselet S, Moerner WE (1999) J Phys Chem A 103(49):10553–10560CrossRefGoogle Scholar
  60. 60.
    Garcia-Parajo MF, Segers-Nolten GMJ, Veerman JA, Greve J, van Hulst NF (2000) Proc Natl Acad Sci USA 97(13):7237–7242CrossRefGoogle Scholar
  61. 61.
    Steinmeyer R, Noskov A, Krasel C, Weber I, Dees C, Harms GS (2005) J Fluoresc 15(5):707–721CrossRefGoogle Scholar
  62. 62.
    McAnaney TB, Zeng W, Doe CFE, Bhanji N, Wakelin S, Pearson DS, Abbyad P, Shi XH, Boxer SG, Bagshaw CR (2005) Biochemistry 44(14):5510–5524CrossRefGoogle Scholar
  63. 63.
    Habuchi S, Cotlet M, Gronheid R, Dirix G, Michiels J, Vanderleyden J, De Schryver FC, Hofkens J (2003) J Am Chem Soc 125(28):8446–8447CrossRefGoogle Scholar
  64. 64.
    Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, Kubota M, Ohashi M, Tsuji FI (1996) Proc Natl Acad Sci USA 93(24):13617–13622CrossRefGoogle Scholar
  65. 65.
    Cinelli RAG, Pellegrini V, Ferrari A, Faraci P, Nifosi R, Tyagi M, Giacca M, Beltram F (2001) Appl Phys Lett 79(20):3353–3355CrossRefGoogle Scholar
  66. 66.
    Chirico G, Diaspro A, Cannone F, Collini M, Bologna S, Pellegrini V, Beltram F (2005) Chemphyschem 6(2):328–335CrossRefGoogle Scholar
  67. 67.
    Stracke F, Blum C, Becker S, Mullen K, Meixner AJ (2004) Chem Phys 300(1–3):153–164CrossRefGoogle Scholar
  68. 68.
    Jung C, Hellriegel C, Michaelis J, Brauchle C (2007) Adv Mater 19(7):956–960CrossRefGoogle Scholar
  69. 69.
    Blum C, Stracke F, Becker S, Mullen K, Meixner AJ (2001) J Phys Chem A 105(29):6983–6990CrossRefGoogle Scholar
  70. 70.
    Xie XS (1996) Acc Chem Res 29(12):598–606CrossRefGoogle Scholar
  71. 71.
    Moerner WE, Orrit M (1999) Science 283(5408):1670–1676CrossRefGoogle Scholar
  72. 72.
    Moerner WE (2002) J Chem Phys 117(24):10925–10937CrossRefGoogle Scholar
  73. 73.
    Jung G, Wiehler J, Gohde W, Tittel J, Basche T, Steipe B, Brauchle C (1998) Bioimaging 6(1):54–61CrossRefGoogle Scholar
  74. 74.
    Cotlet M, Hofkens J, Kohn F, Michiels J, Dirix G, Van Guyse M, Vanderleyden J, De Schryver FC (2001) Chem Phys Lett 336(5–6):415–423CrossRefGoogle Scholar
  75. 75.
    Lounis B, Deich J, Rosell FI, Boxer SG, Moerner WE (2001) J Phys Chem B 105(21):5048–5054CrossRefGoogle Scholar
  76. 76.
    Garcia-Parajo MF, Koopman M, van Dijk E, Subramaniam V, van Hulst NF (2001) Proc Natl Acad Sci USA 98(25):14392–14397CrossRefGoogle Scholar
  77. 77.
    Cotlet M, Hofkens J, Habuchi S, Dirix G, Van Guyse M, Michiels J, Vanderleyden J, De Schryver FC (2001) Proc Natl Acad Sci USA 98(25):14398–14403CrossRefGoogle Scholar
  78. 78.
    Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW (2000) Proc Natl Acad Sci USA 97(22):11996–12001CrossRefGoogle Scholar
  79. 79.
    Baird GS, Zacharias DA, Tsien RY (2000) Proc Natl Acad Sci USA 97(22):11984–11989CrossRefGoogle Scholar
  80. 80.
    Creemers TMH, Lock AJ, Subramaniam V, Jovin TM, Volker S (1999) Nat Struct Biol 6(6):557–560CrossRefGoogle Scholar
  81. 81.
    Creemers TMH, Lock AJ, Subramaniam V, Jovin TM, Volker S (2002) Chem Phys 275(1–3):109–121CrossRefGoogle Scholar
  82. 82.
    Tsutsui H, Karasawa S, Shimizu H, Nukina N, Miyawaki A (2005) EMBO Rep 6(3):233–238CrossRefGoogle Scholar
  83. 83.
    Lessard GA, Habuchi S, Werner JH, Goodwin PM, De Schryver F, Hofkens J, Cotlet M (2008) J Biomed Opt 13(3):031212CrossRefGoogle Scholar
  84. 84.
    Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU (2004) Proc Natl Acad Sci USA 101(45):15905–15910CrossRefGoogle Scholar
  85. 85.
    Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ (2001) Proc Natl Acad Sci USA 98(2):462–467CrossRefGoogle Scholar
  86. 86.
    Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY (2000) Proc Natl Acad Sci USA 97(22):11990–11995CrossRefGoogle Scholar
  87. 87.
    Malvezzi-Campeggi F, Jahnz M, Heinze KG, Dittrich P, Schwille P (2001) Biophys J 81(3):1776–1785CrossRefGoogle Scholar
  88. 88.
    Blum C, Meixner AJ, Subramaniam V (2008) Chemphyschem 9(2):310–315CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  1. 1.Nanobiophysics, MESA+ Institute for Nanotechnology, Faculty of Science and TechnologyUniversity of TwenteAE EnschedeThe Netherlands

Personalised recommendations