Advertisement

Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family

  • G. Ulrich NienhausEmail author
  • Karin Nienhaus
  • Jörg Wiedenmann
Chapter
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)

Abstract

Abstract

GFP-like proteins, originally cloned from marine animals, are genetically encoded fluorescence markers that have become indispensable tools for the life sciences. The search for GFP-like proteins with novel and improved properties is ongoing, driven by the persistent need for advanced and specialized fluorescence labels for cellular imaging. The 3D structures of these proteins are overall similar. However, considerable variations have been found in the covalent structures and the stereochemistry of the chromophore, which govern essential optical properties such as the absorption/emission wavelengths. A detailed understanding of the structure and dynamics of GFP-like proteins greatly aids in the rational engineering of advanced fluorescence marker proteins. In this chapter, we summarize the present knowledge of the structural diversity of GFP-like proteins and discuss how structure and dynamics govern their optical properties.

Graphical Abstract

Keywords

Chromophore Fluorescent protein GFP 

Notes

Acknowledgments

G.U.N. was supported by the Deutsche Forschungsgemeinschaft (DFG) and the State of Baden-Württemberg through the Center for Functional Nanostructures (CFN), by DFG grant Ni 291/9 and by the Baden-Württemberg Stiftung. J.W. acknowledges funding by DFG grant Wi 1990/2, the Network Fluorescence Applications in Biotechnology and Life Sciences, FABLS, Australia, the Landesstiftung Baden-Württemberg (Elite Postdoc Program); the Natural Environment Research Council, UK (NE/G009643/1) and the University of Southampton.

References

  1. 1.
    Nienhaus GU (2008) The green fluorescent protein: a key tool to study chemical processes in living cells. Angew Chem Int Ed Engl 47:8992–8994CrossRefGoogle Scholar
  2. 2.
    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 59:223–239CrossRefGoogle Scholar
  3. 3.
    Chalfie M, Tu Y, Euskirchen G et al (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805CrossRefGoogle Scholar
  4. 4.
    Prasher DC, Eckenrode VK, Ward WW et al (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233CrossRefGoogle Scholar
  5. 5.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefGoogle Scholar
  6. 6.
    Matz MV, Fradkov AF, Labas YA et al (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973CrossRefGoogle Scholar
  7. 7.
    Wiedenmann J, Elke C, Spindler KD et al (2000) Cracks in the beta -can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). Proc Natl Acad Sci USA 97:14091–14096CrossRefGoogle Scholar
  8. 8.
    Wiedenmann J (1997) Die Anwendung eines fluoreszierenden Proteins und weiterer farbiger Proteine und der zugehörigen Gene aus der Artengruppe Anemonia sp. (sulcata) Pennant, (Cnidaria, Anthozoa, Actinaria) in Gentechnologie und Molekularbiologie. Offenlegungsschrift DE 197 18 640 A1, Deutsches Patent- und Markenamt, pp 1–18. In, Offenlegungsschrift DE 197 18 640 A1: Deutsches Patent- und Markenamt; 1997:1–18Google Scholar
  9. 9.
    Wiedenmann J, Nienhaus GU (2006) Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family. Expert Rev Proteomics 3:361–374CrossRefGoogle Scholar
  10. 10.
    Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877CrossRefGoogle Scholar
  11. 11.
    Habuchi S, Ando R, Dedecker P et al (2005) Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc Natl Acad Sci U S A 102:9511–9516CrossRefGoogle Scholar
  12. 12.
    Adam V, Lelimousin M, Boehme S et al (2008) Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc Natl Acad Sci U S A 105:18343–18348CrossRefGoogle Scholar
  13. 13.
    Ormö M, Cubitt AB, Kallio K et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395CrossRefGoogle Scholar
  14. 14.
    Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251CrossRefGoogle Scholar
  15. 15.
    Kummer AD, Kompa C, Niwa H et al (2002) Viscosity-dependent fluorescence decay of the GFP chromophore in solution due to fast internal conversion. J Phys Chem B 106:7554–7559CrossRefGoogle Scholar
  16. 16.
    Voityuk AA, Michel-Beyerle ME, Rösch N (1998) Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins. Chem Phys Lett 296:269–276CrossRefGoogle Scholar
  17. 17.
    Pouwels LJ, Zhang L, Chan NH et al (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
  18. 18.
    Katranidis A, Atta D, Schlesinger R et al (2009) Fast biosynthesis of GFP molecules: a single-molecule fluorescence study. Angew Chem Int ed 48:1758–1761CrossRefGoogle Scholar
  19. 19.
    Youvan DC, Michel-Beyerle ME (1996) Structure and fluorescence mechanism of GFP. Nat Biotechnol 14:1219–1220CrossRefGoogle Scholar
  20. 20.
    Nienhaus GU (2010) The “wiggling and jiggling of atoms” leading to excited-state proton transfer in green fluorescent protein. ChemPhysChem 11:971–974Google Scholar
  21. 21.
    Lossau H, Kummer A, Heinecke R et al (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
  22. 22.
    Gross LA, Baird GS, Hoffman RC et al (2000) The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97:11990–11995CrossRefGoogle Scholar
  23. 23.
    Wiedenmann J, Schenk A, Röcker C et al (2002) A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc Natl Acad Sci USA 99:11646–11651CrossRefGoogle Scholar
  24. 24.
    Nienhaus GU, Wiedenmann J (2009) Structure, dynamics and optical properties of fluorescent proteins: perspectives for marker development. ChemPhysChem 10:1369–1379CrossRefGoogle Scholar
  25. 25.
    Abbyad P, Childs W, Shi X et al (2007) Dynamic Stokes shift in green fluorescent protein variants. Proc Natl Acad Sci U S A 104:20189–20194CrossRefGoogle Scholar
  26. 26.
    Shu X, Shaner NC, Yarbrough CA et al (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45:9639–9646CrossRefGoogle Scholar
  27. 27.
    Remington SJ, Wachter RM, Yarbrough DK et al (2005) zFP538, a yellow-fluorescent protein from Zoanthus, contains a novel three-ring chromophore. Biochemistry 44:202–212CrossRefGoogle Scholar
  28. 28.
    Lukyanov KA, Fradkov AF, Gurskaya NG et al (2000) Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J Biol Chem 275:25879–25882CrossRefGoogle Scholar
  29. 29.
    Quillin ML, Anstrom DM, Shu X et al (2005) Kindling fluorescent protein from Anemonia sulcata: dark-state structure at 1.38 A resolution. Biochemistry 44:5774–5787CrossRefGoogle Scholar
  30. 30.
    Wilmann PG, Petersen J, Devenish RJ et al (2005) Variations on the GFP chromophore: a polypeptide fragmentation within the chromophore revealed in the 2.1-A crystal structure of a nonfluorescent chromoprotein from Anemonia sulcata. J Biol Chem 280:2401–2404CrossRefGoogle Scholar
  31. 31.
    Andresen M, Wahl MC, Stiel AC et al (2005) Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci U S A 102:13070–13074CrossRefGoogle Scholar
  32. 32.
    Phillips GN Jr (1997) Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol 7:821–827CrossRefGoogle Scholar
  33. 33.
    Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97:11984–11989CrossRefGoogle Scholar
  34. 34.
    Vrzheshch PV, Akovbian NA, Varfolomeyev SD et al (2000) Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions. FEBS Lett 487:203–208CrossRefGoogle Scholar
  35. 35.
    Alieva NO, Konzen KA, Field SF et al (2008) Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680CrossRefGoogle Scholar
  36. 36.
    Gurskaya NG, Fradkov AF, Terskikh A et al (2001) GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett 507:16–20CrossRefGoogle Scholar
  37. 37.
    Verkhusha VV, Lukyanov KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22:289–296CrossRefGoogle Scholar
  38. 38.
    Yarbrough D, Wachter RM, Kallio K et al (2001) Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution. Proc Natl Acad Sci U S A 98:462–467CrossRefGoogle Scholar
  39. 39.
    Yang TT, Sinai P, Green G et al (1998) Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J Biol Chem 273:8212–8216CrossRefGoogle Scholar
  40. 40.
    Cubitt AB, Heim R, Adams SR et al (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20:448–455CrossRefGoogle Scholar
  41. 41.
    Pedelacq JD, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88CrossRefGoogle Scholar
  42. 42.
    Kredel S, Nienhaus K, Wolff M et al (2008) Optimized and far-red emitting variants of fluorescent protein eqFP611. Chem Biol 15:224–233CrossRefGoogle Scholar
  43. 43.
    Fuchs J, Böhme S, Oswald F et al (2010) Imaging protein movements in live cells with super-resolution using mIrisFP. Nat Methods 7:627–630CrossRefGoogle Scholar
  44. 44.
    Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med (Berlin, Germany) 81:678–699Google Scholar
  45. 45.
    Link CD, Fonte V, Hiester B et al (2006) Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide. J Biol Chem 281:1808–1816CrossRefGoogle Scholar
  46. 46.
    Wiedenmann J, Oswald F, Nienhaus GU (2009) Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB life 61:1029–1042CrossRefGoogle Scholar
  47. 47.
    Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721CrossRefGoogle Scholar
  48. 48.
    Bulina ME, Chudakov DM, Britanova OV et al (2006) A genetically encoded photosensitizer. Nat Biotechnol 24:95–99CrossRefGoogle Scholar
  49. 49.
    Tour O, Meijer RM, Zacharias DA et al (2003) Genetically targeted chromophore-assisted light inactivation. Nat Biotechnol 21:1505–1508CrossRefGoogle Scholar
  50. 50.
    Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921CrossRefGoogle Scholar
  51. 51.
    Zacharias DA, Violin JD, Newton AC et al (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916CrossRefGoogle Scholar
  52. 52.
    Wiedenmann J, Ivanchenko S, Oswald F et al (2004) EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101:15905–15910CrossRefGoogle Scholar
  53. 53.
    Wiedenmann J, Vallone B, Renzi F et al (2005) The red fluorescent protein eqFP611 and its genetically engineered dimeric variants. J Biomed Optics 10:014003 (014007 pages)CrossRefGoogle Scholar
  54. 54.
    Campbell RE, Tour O, Palmer AE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882CrossRefGoogle Scholar
  55. 55.
    Kredel S, Oswald F, Nienhaus K et al (2009) mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures. PLoS One 4:e4391CrossRefGoogle Scholar
  56. 56.
    Fradkov AF, Verkhusha VV, Staroverov DB et al (2002) Far-red fluorescent tag for protein labelling. Biochem J 368:17–21CrossRefGoogle Scholar
  57. 57.
    Nienhaus GU, Nienhaus K, Hölzle A et al (2006) Photoconvertible fluorescent protein EosFP-biophysical properties and cell biology applications. Photochem Photobiol 82:351–358CrossRefGoogle Scholar
  58. 58.
    Bulina ME, Verkhusha VV, Staroverov DB et al (2003) Hetero-oligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins. Biochem J 371:109–114CrossRefGoogle Scholar
  59. 59.
    Shaner NC, Lin MZ, McKeown MR et al (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551CrossRefGoogle Scholar
  60. 60.
    Ai HW, Henderson JN, Remington SJ et al (2006) Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem J 400:531–540CrossRefGoogle Scholar
  61. 61.
    van Thor JJ, Gensch T, Hellingwerf KJ et al (2002) Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222. Nat Struct Biol 9:37–41CrossRefGoogle Scholar
  62. 62.
    Ai HW, Shaner NC, Cheng Z et al (2007) Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46:5904–5910CrossRefGoogle Scholar
  63. 63.
    Adam V, Nienhaus K, Bourgeois D et al (2009) Structural basis of enhanced photoconversion yield in green fluorescent protein-like protein Dendra2. Biochemistry 48:4905–4915CrossRefGoogle Scholar
  64. 64.
    Shcherbo D, Merzlyak EM, Chepurnykh TV et al (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4:741–746CrossRefGoogle Scholar
  65. 65.
    Piatkevich KD, Hulit J, Subach OM et al (2010) Monomeric red fluorescent proteins with a large Stokes shift. Proc Natl Acad Sci U S A 107:5369–5374CrossRefGoogle Scholar
  66. 66.
    Wachter RM, Elsliger MA, Kallio K et al (1998) Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. Structure 6:1267–1277CrossRefGoogle Scholar
  67. 67.
    Shagin DA, Barsova EV, Yanushevich YG et al (2004) GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol Biol Evol 21:841–850CrossRefGoogle Scholar
  68. 68.
    Petersen J, Wilmann PG, Beddoe T et al (2003) The 2.0-Å crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. J Biol Chem 278:44626–44631CrossRefGoogle Scholar
  69. 69.
    Loos DC, Habuchi S, Flors C et al (2006) Photoconversion in the red fluorescent protein from the sea anemone Entacmaea quadricolor: is cis-trans isomerization involved? J Am Chem Soc 128:6270–6271CrossRefGoogle Scholar
  70. 70.
    Nienhaus K, Nar H, Heilker R et al (2008) Trans-cis isomerization is responsible for the red-shifted fluorescence in variants of the red fluorescent protein eqFP611. J Am Chem Soc 130:12578–12579CrossRefGoogle Scholar
  71. 71.
    Terskikh A, Fradkov A, Ermakova G et al (2000) “Fluorescent timer”: protein that changes color with time. Science 290:1585–1588CrossRefGoogle Scholar
  72. 72.
    Subach FV, Subach OM, Gundorov IS et al (2009) Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat Chem Biol 5:118–126CrossRefGoogle Scholar
  73. 73.
    Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A 91:12501–12504CrossRefGoogle Scholar
  74. 74.
    Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182CrossRefGoogle Scholar
  75. 75.
    Kajihara D, Hohsaka T, Sisido M (2005) Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position. Protein Eng Des Sel 18:273–278CrossRefGoogle Scholar
  76. 76.
    Goulding A, Shrestha S, Dria K et al (2008) Red fluorescent protein variants with incorporated non-natural amino acid analogues. Protein Eng Des Sel 21:101–106CrossRefGoogle Scholar
  77. 77.
    Subach OM, Gundorov IS, Yoshimura M et al (2008) Conversion of red fluorescent protein into a bright blue probe. Chem Biol 15:1116–1124CrossRefGoogle Scholar
  78. 78.
    Subach OM, Malashkevich VN, Zencheck WD et al (2010) Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins. Chem Biol 17:333–341CrossRefGoogle Scholar
  79. 79.
    Henderson JN, Gepshtein R, Heenan JR et al (2009) Structure and mechanism of the photoactivatable green fluorescent protein. J Am Chem Soc 131:4176–4177CrossRefGoogle Scholar
  80. 80.
    Ando R, Hama H, Yamamoto-Hino M et al (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
  81. 81.
    Nienhaus K, Nienhaus GU, Wiedenmann J et al (2005) Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP. Proc Natl Acad Sci U S A 102:9156–9159CrossRefGoogle Scholar
  82. 82.
    Gurskaya NG, Verkhusha VV, Shcheglov AS et al (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nat Biotechnol 24:461–465CrossRefGoogle Scholar
  83. 83.
    Labas YA, Gurskaya NG, Yanushevich YG et al (2002) Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci U S A 99:4256–4261CrossRefGoogle Scholar
  84. 84.
    Oswald F, Schmitt F, Leutenegger A et al (2007) Contributions of host and symbiont pigments to the coloration of reef corals. FEBS J 274:1102–1109CrossRefGoogle Scholar
  85. 85.
    Mizuno H, Mal TK, Tong KI et al (2003) Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Mol Cell 12:1051–1058CrossRefGoogle Scholar
  86. 86.
    Hayashi I, Mizuno H, Tong KI et al (2007) Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede. J Mol Biol 372:918–926CrossRefGoogle Scholar
  87. 87.
    Lelimousin M, Adam V, Nienhaus GU et al (2009) Photoconversion of the fluorescent protein EosFP: a hybrid potential simulation study reveals intersystem crossings. J Am Chem Soc 131:16814–16823CrossRefGoogle Scholar
  88. 88.
    Lukyanov KA, Chudakov DM, Lukyanov S et al (2005) Innovation: photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891CrossRefGoogle Scholar
  89. 89.
    Chudakov DM, Belousov VV, Zaraisky AG et al (2003) Kindling fluorescent proteins for precise in vivo photolabeling. Nat Biotechnol 21:191–194CrossRefGoogle Scholar
  90. 90.
    Wilmann PG, Turcic K, Battad JM et al (2006) The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein. J Mol Biol 364:213–224CrossRefGoogle Scholar
  91. 91.
    Andresen M, Stiel AC, Trowitzsch S et al (2007) Structural basis for reversible photoswitching in Dronpa. Proc Natl Acad Sci U S A 104:13005–13009CrossRefGoogle Scholar
  92. 92.
    Henderson JN, Ai HW, Campbell RE et al (2007) Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc Natl Acad Sci U S A 104:6672–6677CrossRefGoogle Scholar
  93. 93.
    Schäfer LV, Groenhof G, Klingen AR et al (2007) Photoswitching of the fluorescent protein asFP595: mechanism, proton pathways, and absorption spectra. Angew Chem Int Ed 46:530–536CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • G. Ulrich Nienhaus
    • 1
    • 2
    Email author
  • Karin Nienhaus
    • 1
  • Jörg Wiedenmann
    • 3
  1. 1.Institute of Applied Physics and Center for Functional NanostructuresKarlsruhe Institute of Technology (KIT)KarlsruheGermany
  2. 2.Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.National Oceanography CentreUniversity of SouthamptonSouthamptonUK

Personalised recommendations