Advertisement

Synthetic Biology of Autofluorescent Proteins

  • Michael Georg Hoesl
  • Lars Merkel
  • Nediljko BudisaEmail author
Chapter
Part of the Springer Series on Fluorescence book series (SS FLUOR, volume 11)

Abstract

Abstract

Autofluorescent proteins (FPs), which to date are predominately used as tools in cell biology and spectroscopy, have arrived in the focus of synthetic biology. Thereby, the intention is to supplement classically used protein design methods such as site-directed mutagenesis or guided evolution by expanding the scope of protein synthesis. This is achieved by the co-translational introduction of novel noncanonical amino acids (NCAAs) into proteins. In the following chapter, we present current applications of an expanded amino acid repertoire for the design of spectral and folding properties of FPs. We will show that NCAAs are not only useful tools to study fundamental aspects of photophysics but also have great potential to generate novel FP tools for cell biology applications. On the one hand, aromatic amino acids other than the naturally occurring His, Tyr, Phe, and Trp were used to create novel spectral classes of FPs by direct chromophore modification. On the other hand, NCAAs were also applied for “FP protein matrix engineering” to influence chromophore fluorescence and overall folding. We also illustrate a practical application of these principles by presenting “golden annexin A5” as a novel apoptosis detection tool designed by synthetic biology methods. Finally, we describe a potential route to convert any protein of interest into a chromo-protein by introduction of novel synthetic autofluorescent amino acids.

Graphical Abstract

Keywords

Chromophore variants ECFP EYFP GFP GFP structure non-canonical amino acid synthetic biology 

References

  1. 1.
    Sample V, Newman RH, Zhang J (2009) The structure and function of fluorescent proteins. Chem Soc Rev 38(10):2852–2864CrossRefGoogle Scholar
  2. 2.
    Stepanenko OV, Verkhusha VV, Kuznetsova IM, Uversky VN, Turoverov KK (2008) Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes. Curr Protein Pept Sci 9(4):338–369CrossRefGoogle Scholar
  3. 3.
    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(16):8362–8367CrossRefGoogle Scholar
  4. 4.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefGoogle Scholar
  5. 5.
    Crameri A, Whitehorn EA, Tate E, Stemmer WPC (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14(3):315–319CrossRefGoogle Scholar
  6. 6.
    Cormack BP, Valdivia RH, Falkow S (1996) Facs-optimized mutants of the green fluorescent protein (gfp). Gene 173(1):33–38CrossRefGoogle Scholar
  7. 7.
    Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6(2):178–182CrossRefGoogle Scholar
  8. 8.
    Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273(5280):1392–1395CrossRefGoogle Scholar
  9. 9.
    Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91(26):12501–12504CrossRefGoogle Scholar
  10. 10.
    Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent anthozoa species. Nat Biotechnol 17(10):969–973CrossRefGoogle Scholar
  11. 11.
    Bae JH, Rubini M, Jung G, Wiegand G, Seifert MHJ, Azim MK, Kim JS, Zumbusch A, Holak TA, Moroder L, Huber R, Budisa N (2003) Expansion of the genetic code enables design of a novel “gold”' class of green fluorescent proteins. J Mol Biol 328(5):1071–1081CrossRefGoogle Scholar
  12. 12.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2(12):905–909CrossRefGoogle Scholar
  13. 13.
    Kwon I, Tirrell DA (2007) Site-specific incorporation of tryptophan analogues into recombinant proteins in bacterial cells. J Am Chem Soc 129(34):10431–10437CrossRefGoogle Scholar
  14. 14.
    Wang L, Xie JM, Deniz AA, Schultz PG (2003) Unnatural amino acid mutagenesis of green fluorescent protein. J Org Chem 68(1):174–176CrossRefGoogle Scholar
  15. 15.
    Budisa N, Pal PP, Alefelder S, Birle P, Krywcun T, Rubini M, Wenger W, Bae JH, Steiner T (2004) Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code. Biol Chem 385(2):191–202CrossRefGoogle Scholar
  16. 16.
    Goulding A, Shrestha S, Dria K, Hunt E, Deo SK (2008) Red fluorescent protein variants with incorporated non-natural amino acid analogues. Protein Eng Des Sel 21(2):101–106CrossRefGoogle Scholar
  17. 17.
    Pal PP, Bae JH, Azim MK, Hess P, Friedrich R, Huber R, Moroder L, Budisa N (2005) Structural and spectral response of Aequorea victoria green fluorescent proteins to chromophore fluorination. Biochemistry 44(10):3663–3672CrossRefGoogle Scholar
  18. 18.
    Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ (2001) Refined crystal structure of dsred, a red fluorescent protein from coral, at 2.0-angstrom resolution. Proc Natl Acad Sci USA 98(2):462–467CrossRefGoogle Scholar
  19. 19.
    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(1–3):1–16CrossRefGoogle Scholar
  20. 20.
    Malo GD, Pouwels LJ, Wang MT, Weichsel A, Montfort WR, Rizzo MA, Piston DW, Wachter RM (2007) X-ray structure of cerulean gfp: A tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry 46(35):9865–9873CrossRefGoogle Scholar
  21. 21.
    Demachy I, Ridard J, Laguitton-Pasquier H, Durnerin E, Vallverdu G, Archirel P, Levy B (2005) Cyan fluorescent protein: molecular dynamics, simulations, and electronic absorption spectrum. J Phys Chem B 109(50):24121–24133CrossRefGoogle Scholar
  22. 22.
    Sinha HK, Dogra SK, Krishnamurthy M (1987) Excited-state and ground-state proton-transfer reactions in 5-aminoindole. Bull Chem Soc Jpn 60(12):4401–4407CrossRefGoogle Scholar
  23. 23.
    Craggs TD (2009) Green fluorescent protein: structure, folding and chromophore maturation. Chem Soc Rev 38(10):2865–2875CrossRefGoogle Scholar
  24. 24.
    Kasha M (1952) Collisional perturbation of spin orbital coupling and the mechanism of fluorescence quenching. A visual demonstration of the perturbation. J Chem Phys 20(1):71–74CrossRefGoogle Scholar
  25. 25.
    Bonnett R, Harriman A, Kozyrev AN (1992) Photophysics of halogenated porphyrins. J Chem Soc Faraday Trans 88(6):763–769CrossRefGoogle Scholar
  26. 26.
    Bondi A (1964) Van der waals volumes and radii. J Phys Chem 68(3):441–451CrossRefGoogle Scholar
  27. 27.
    Brooks B, Phillips RS, Benisek WF (1998) High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of delta(5)-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics. Biochemistry 37(27):9738–9742CrossRefGoogle Scholar
  28. 28.
    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(6):273–278CrossRefGoogle Scholar
  29. 29.
    Budisa N, Alefelder S, Bae JH, Golbik R, Minks C, Huber R, Moroder L (2001) Proteins with beta-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids. Protein Sci 10(7):1281–1292CrossRefGoogle Scholar
  30. 30.
    Visser NV, Borst JW, Hink MA, van Hoek A, Visser AJWG (2005) Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophys Chem 116(3):207–212CrossRefGoogle Scholar
  31. 31.
    Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein – mechanism and applications. J Biol Chem 276(31):29188–29194CrossRefGoogle Scholar
  32. 32.
    Budisa N (2003) Expression of “tailor-made” proteins via incorporation of synthetic amino acids by using cell-free protein synthesis. Cell-free protein expression. Springer-Verlag, HeidelbergGoogle Scholar
  33. 33.
    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(24):13617–13622CrossRefGoogle Scholar
  34. 34.
    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(15):4423–4431CrossRefGoogle Scholar
  35. 35.
    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(1–2):183–193CrossRefGoogle Scholar
  36. 36.
    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(19):4791–4798CrossRefGoogle Scholar
  37. 37.
    Palm GJ, Wlodawer A (1999) Spectral variants of green fluorescent protein. In: Green fluorescent protein, vol 302 (Ed. Conn P M). Methods in enzymology. Academic, San Diego, pp 378–394Google Scholar
  38. 38.
    Prendergast FG (1999) Biophysics of the green fluorescent protein. Methods Cell Biol 58:1–18CrossRefGoogle Scholar
  39. 39.
    Budisa N, Pipitone O, Siwanowicz I, Rubini M, Pal PP, Holak TA, Gelmi ML (2004) Efforts towards the design of ‘teflon’ proteins: in vivo translation with trifluorinated leucine and methionine analogues. Chem Biodivers 1(10):1465–1475CrossRefGoogle Scholar
  40. 40.
    Houston ME, Harvath L, Honek JF (1997) Synthesis of and chemotactic responses elicited by fmet-leu-phe analogs containing difluoro- and trifluoromethionine. Bioorg Med Chem Lett 7(23):3007–3012CrossRefGoogle Scholar
  41. 41.
    Yoo TH, Tirrell DA (2007) High-throughput screening for methionyl-trna synthetases that enable residue-specific incorporation of noncanonical amino acids into recombinant proteins in bacterial cells. Angew Chem Int Ed 46(28):5340–5343CrossRefGoogle Scholar
  42. 42.
    O'Hagan D, Rzepa HS (1997) Some influences of fluorine in bioorganic chemistry. Chem Commun 7:645–652CrossRefGoogle Scholar
  43. 43.
    Yoo TH, Link AJ, Tirrell DA (2007) Evolution of a fluorinated green fluorescent protein. Proc Natl Acad Sci USA 104(35):13887–13890CrossRefGoogle Scholar
  44. 44.
    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):87–90CrossRefGoogle Scholar
  45. 45.
    Steiner T, Hess P, Bae JH, Wiltschi B, Moroder L, Budisa N (2008) Synthetic biology of proteins: tuning gfps folding and stability with fluoroproline. PLoS ONE 3(2):e1680CrossRefGoogle Scholar
  46. 46.
    Wedemeyer WJ, Welker E, Scheraga HA (2002) Proline cis-trans isomerization and protein folding. Biochemistry 41(50):14637–14644CrossRefGoogle Scholar
  47. 47.
    Dugave C, Demange L (2003) Cis-trans isomerization of organic molecules and biomolecules: implications and applications. Chem Rev 103(7):2475–2532CrossRefGoogle Scholar
  48. 48.
    Pal D, Chakrabarti P (1999) Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations. J Mol Biol 294(1):271–288CrossRefGoogle Scholar
  49. 49.
    Milner-White EJ, Bell LH, Maccallum PH (1992) Pyrrolidine ring puckering in cis and trans-proline residues in proteins and polypeptides. Different puckers are favoured in certain situations. J Mol Biol 228(3):725–734CrossRefGoogle Scholar
  50. 50.
    Renner C, Alefelder S, Bae JH, Budisa N, Huber R, Moroder L (2001) Fluoroprolines as tools for protein design and engineering. Angew Chem Int Ed 40(5):923–925CrossRefGoogle Scholar
  51. 51.
    Holmgren SK, Taylor KM, Bretscher LE, Raines RT (1998) Code for collagen's stability deciphered. Nature 392(6677):666–667CrossRefGoogle Scholar
  52. 52.
    Holmgren SK, Bretscher LE, Taylor KM, Raines RT (1999) A hyperstable collagen mimic. Chem Biol 6(2):63–70CrossRefGoogle Scholar
  53. 53.
    Eberhardt ES, Panasik N, Raines RT (1996) Inductive effects on the energetics of prolyl peptide bond isomerization: implications for collagen folding and stability. J Am Chem Soc 118(49):12261–12266CrossRefGoogle Scholar
  54. 54.
    Moroder L, Budisa N (2010) Synthetic biology of protein folding. Chem Phys Chem 11(6):1181–1187CrossRefGoogle Scholar
  55. 55.
    Budisa N (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew Chem Int Ed 43(47):6426–6463CrossRefGoogle Scholar
  56. 56.
    Xiao GY, Parsons JF, Tesh K, Armstrong RN, Gilliland GL (1998) Conformational changes in the crystal structure of rat glutathione transferase m1-1 with global substitution of 3-fluorotyrosine for tyrosine. J Mol Biol 281(2):323–339CrossRefGoogle Scholar
  57. 57.
    Bae JH, Pal PP, Moroder L, Huber R, Budisa N (2004) Crystallographic evidence for isomeric chromophores in 3-fluorotyrosyl-green fluorescent protein. Chem Bio Chem 5(5):720–722CrossRefGoogle Scholar
  58. 58.
    Seifert MH, Ksiazek D, Azim MK, Smialowski P, Budisa N, Holak TA (2002) Slow exchange in the chromophore of a green fluorescent protein variant. J Am Chem Soc 124(27):7932–7942CrossRefGoogle Scholar
  59. 59.
    Hoesl MG, Larregola M, Cui H, Budisa N (2010) Azatryptophans as tools to study polarity requirements for folding of green fluorescent protein. J Pept Sci 16(10):589–595Google Scholar
  60. 60.
    Andrews BT, Schoenfish AR, Roy M, Waldo G, Jennings PA (2007) The rough energy landscape of superfolder gfp is linked to the chromophore. J Mol Biol 373:476–490CrossRefGoogle Scholar
  61. 61.
    Barondeau DP, Putnam CD, Kassmann CJ, Tainer JA, Getzoff ED (2003) Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures. Proc Natl Acad Sci USA 100(21):12111–12116CrossRefGoogle Scholar
  62. 62.
    Reid BG, Flynn GC (1997) Chromophore formation in green fluorescent protein. Biochemistry 36(22):6786–6791CrossRefGoogle Scholar
  63. 63.
    Ward WW, Prentice HJ, Roth AF, Cody CW, Reeves SC (1982) Spectral perturbations of mutants of recombinant Aequorea victoria green-fluorescent protein. Photochem Photobiol 35(6):803–808CrossRefGoogle Scholar
  64. 64.
    Chen MC, Lambert CR, Urgitis JD, Zimmer M (2001) Photoisomerization of green fluorescent protein and the dimensions of the chromophore cavity. Chem Phys 270(1):157–164CrossRefGoogle Scholar
  65. 65.
    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(10):1267–1277CrossRefGoogle Scholar
  66. 66.
    Villoing A, Ridhoir M, Cinquin B, Erard M, Alvarez L, Vallverdu G, Pernot P, Grailhe R, Merola F, Pasquier H (2008) Complex fluorescence of the cyan fluorescent protein: comparisons with the h148d variant and consequences for quantitative cell imaging. Biochemistry 47(47):12483–12492CrossRefGoogle Scholar
  67. 67.
    Reid KSC, Lindley PF, Thornton JM (1985) Sulfur-aromatic interactions in proteins. FEBS Lett 190(2):209–213CrossRefGoogle Scholar
  68. 68.
    Viguera AR, Serrano L (1995) Side-chain interactions between sulfur-containing amino acids and phenyalanine in alpha-helices. Biochemistry 34(27):8771–8779CrossRefGoogle Scholar
  69. 69.
    Bae JH, Alefelder S, Kaiser JT, Friedrich R, Moroder L, Huber R, Budisa N (2001) Incorporation of beta-selenolo 3, 2-b pyrrolyl-alanine into proteins for phase determination in protein x-ray crystallography. J Mol Biol 309(4):925–936CrossRefGoogle Scholar
  70. 70.
    Kurschus FC, Pal PP, Baumler P, Jenne DE, Wiltschi B, Budisa N (2009) Gold fluorescent annexin a5 as a novel apoptosis detection tool. Cytom A 75A(7):626–633CrossRefGoogle Scholar
  71. 71.
    Ernst JD, Mall A, Chew G (1994) Annexins possess functionally distinguishable ca2+ and phospholipid binding domains. Biochem Biophys Res Commun 200(2):867–876CrossRefGoogle Scholar
  72. 72.
    Ernst JD, Yang L, Rosales JL, Broaddus VC (1998) Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells. Anal Biochem 260(1):18–23CrossRefGoogle Scholar
  73. 73.
    Kuhn SM, Rubini M, Mueller MA, Skerra A (2011) Biosynthesis of a Fluorescent Protein with Extreme Pseudo-Stokes Shift by Introducing a Genetically Encoded Non-Natural Amino Acid outside the Fluorophore. J Am Chem Soc 133:3708–3711CrossRefGoogle Scholar
  74. 74.
    Lepthien S, Hoesl MG, Merkel L, Budisa N (2008) Azatryptophans endow proteins with intrinsic blue fluorescence. Proc Natl Acad Sci USA 105(42):16095–16100CrossRefGoogle Scholar
  75. 75.
    Merkel L, Hoesl MG, Albrecht M, Schmidt A, Budisa N (2010) Blue fluorescent amino acids as in vivo building blocks for proteins. Chem Bio Chem 11(3):305–314CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2011

Authors and Affiliations

  • Michael Georg Hoesl
    • 1
  • Lars Merkel
    • 1
  • Nediljko Budisa
    • 1
    Email author
  1. 1.Department of ChemistryBerlin Institute of Technology/TU Berlin, Biocatalysis GroupBerlinGermany

Personalised recommendations