Designing novel spectral classes of proteins with a tryptophan-expanded genetic code

Nediljko Budisa 1.  and Prajna Paramita Pal 2.
  • 1. Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany
  • 2. Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany

Abstract

Fluorescence methods are now well-established and powerful tools to study biological macromolecules. The canonical amino acid tryptophan (Trp), encoded by a single UGG triplet, is the main reporter of intrinsic fluorescence properties of most natural proteins and peptides and is thus an attractive target for tailoring their spectral properties. Recent advances in research have provided substantial evidence that the natural protein translational machinery can be genetically reprogrammed to introduce a large number of non-coded (i.e. noncanonical) Trp analogues and surrogates into various proteins. Especially attractive targets for such an engineering approach are fluorescent proteins in which the chromophore is formed post-translationally from an amino acid sequence, like the green fluorescent protein from Aequorea victoria. With the currently available translationally active fluoro-, hydroxy-, amino-, halogen-, and chalcogen-containing Trp analogues and surrogates, the traditional methods for protein engineering and design can be supplemented or even fully replaced by these novel approaches. Future research will provide a further increase in the number of Trp-like amino acids that are available for redesign (by engineering of the genetic code) of native Trp residues and enable novel strategies to generate proteins with tailored spectral properties.

  • Bae, J.H., Alefelder, S., Kaiser, J.T., Friedrich, R., Moroder, L., Huber, R. and Budisa, N. (2001). Incorporation of b-selenolo 3,2-b pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography. J. Mol. Biol. 309, 925–936.

    • Google Scholar
    • Export Citation
  • Bae, J.H., Rubini, M., Jung, G., Wiegand, G., Seifert, M.H.J., Azim, M.K., Kim, J.S., Zumbusch, A., Holak, T.A., Moroder, L., Huber, R. and Budisa, N. (2003). Expansion of the genetic code enables design of a novel ‘gold’ class of green fluorescent proteins. J. Mol. Biol. 328, 1071–1081.

    • Google Scholar
    • Export Citation
  • Brawerman, G. and Ycas, M. (1957). Incorporation of the amino acid analog tryptazan into the protein of Escherichia coli. Arch. Biochem. Biophys. 68, 112–117.

    • Google Scholar
    • Export Citation
  • Bronskill, P.M. and Wong, J.T.F. (1988). Suppression of fluorescence of tryptophan residues in proteins by replacement with 4-fluorotryptophan. Biochem. J. 249, 305–308.

    • Google Scholar
    • Export Citation
  • Budisa, N. (2003). Expression of ‘tailor-made’ proteins via incorporation of synthetic amino acids by using cell-free protein synthesis. In: Cell Free Protein Expression, J.R. Swartz, ed. (Berlin, Heidelberg, New York: Springer Verlag), pp. 89–98.

  • Budisa, N. (2004a). Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew. Chem. 45, in press.

  • Budisa, N. (2004b). Protein engineering and design with an expanded amino acid repertoire. Technical University Munich, Germany.

  • Budisa, N., Minks, C., Medrano, F.J., Lutz, J., Huber, R. and Moroder, L. (1998). Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V. Proc. Natl. Acad. Sci. USA 95, 455–459.

    • Google Scholar
    • Export Citation
  • Budisa, N., Minks, C., Alefelder, S., Wenger, W., Dong, F.M., Moroder, L. and Huber, R. (1999a). Toward the experimental codon reassignment in vivo: protein building with an expanded amino acid repertoire. FASEB J. 13, 41–51.

    • Google Scholar
    • Export Citation
  • Budisa, N., Moroder, L. and Huber, R. (1999b). Structure and evolution of the genetic code viewed from the perspective of the experimentally expanded amino acid repertoire in vivo. Cell. Mol. Life Sci. 55, 1626–1635.

    • Google Scholar
    • Export Citation
  • Budisa, N., Alefelder, S., Bae, J.H., Golbik, R., Minks, C., Huber, R. and Moroder, L. (2001). Proteins with b-(thienopyrrolyl) alanines as alternative chromophores and pharmaceutically active amino acids. Protein Sci. 10, 1281–1292.

    • Google Scholar
    • Export Citation
  • Budisa, N., Rubini, M., Bae, J.H., Weyher, E.,Wenger,W., Golbik, R., Huber, R. and Moroder, L. (2002). Global replacement of tryptophan with aminotryptophans generates non-invasive protein-based optical pH sensors. Angew. Chem. Int. Ed. 41, 4066–4069.

    • Google Scholar
    • Export Citation
  • Budisa, N., Pal, P.P., Alefelder, S., Birle, P., Krywcun, T., Rubini, M., Wenger, W., Bae, J.H. and Steiner, T. (2004). Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code. Biol. Chem. 385, 191–202.

    • Google Scholar
    • Export Citation
  • Burley, S.K. and Petsko, G.A. (1988). Weakly polar interactions in proteins. Adv. Protein Chem. 39, 125–189.

    • Google Scholar
    • Export Citation
  • Chapeville, F., Ehrenstein, G.V., Benzer, S., Weisblum, B., Ray, W.J. and Lipmann, F. (1962). On role of soluble ribonucleic acid in coding for amino acids. Proc. Natl. Acad. Sci. USA 48, 1086–1098.

    • Google Scholar
    • Export Citation
  • Crick, F.H.C. (1957). On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163.

  • Dayhoff, M.O. (1972). Atlas of protein sequence and structure (Washington DC, USA: National Biomedical Research Foundation).

  • De Filippis, V., De Boni, S., De Dea, E., Dalzoppo, D., Grandi, C. and Fontana, A. (2004). Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1–47 of hirudin as a probe of hirudin folding and thrombin recognition. Protein Sci. 13, 1489–1502.

    • Google Scholar
    • Export Citation
  • Deming, T.J., Fournier, M.J., Mason, T.L. and Tirrell, D.A. (1996). Structural modification of a periodic polypeptide through biosynthetic replacement of proline with azetidine-2-carboxylic acid. Macromolecules 29, 1442–1444.

    • Google Scholar
    • Export Citation
  • Doublie, S., Bricogne, G., Gilmore, C. and Carter, C.W. (1995). Tryptophanyl-transfer-RNA synthetase crystal-structure reveals an unexpected homology to tyrosyl-transfer-RNA synthetase. Structure 3, 17–31.

    • Google Scholar
    • Export Citation
  • Dougherty, D.A. (1996). Cation-p interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science 271, 163–168.

    • Google Scholar
    • Export Citation
  • Evans, C.S. and Bell, E.A. (1980). Neuroactive plant amino acids and amines. Trends Neurosci. 3, 70–72.

  • Golbik, R., Fischer, G. and Fersht, A.R. (1999). Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl-prolyl cis/ trans isomerization by human cytosolic cyclophilin (Cyp18). Protein Sci. 8, 1505–1514.

    • Google Scholar
    • Export Citation
  • Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. and Tsien, R.Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein-mechanism and applications. J. Biol. Chem. 276, 29188–29194.

    • Google Scholar
    • Export Citation
  • Hamano-Takaku, F., Iwama, T., Saito-Yano, S., Takaku, K., Monden, Y., Kitabatake, M., Soll, D. and Nishimura, S. (2000). A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine. J. Biol. Chem. 275, 40324–40328.

    • Google Scholar
    • Export Citation
  • Hohsaka, T. and Sisido, M. (2002). Incorporation of non-natural amino acids into proteins. Curr. Opin. Chem. Biol. 6, 809–815.

    • Google Scholar
    • Export Citation
  • Hortin, G. and Boime, I. (1983). Applications of amino acid analogs for studying co-translational and posttranslational modifications of proteins. Methods Enzymol. 96, 777–784.

    • Google Scholar
    • Export Citation
  • Kiick, K.L., van Hest, J.C.M. and Tirrell, D.A. (2000). Expanding the scope of protein biosynthesis by altering the methionyl-tRNA synthetase activity of a bacterial expression host. Angew. Chem. Int. Ed. 39, 2148–2151.

    • Google Scholar
    • Export Citation
  • Kishi, T., Tanaka, M. and Tanaka, J. (1977). Electronic absorption and fluorescence-spectra of 5-hydroxytryptamine (serotonin)-protonation in excited-state. Bull. Chem. Soc. Jpn. 50, 1267–1271.

    • Google Scholar
    • Export Citation
  • Lakowitz, J.R. (1999). Protein fluorescence (New York, USA: Kluwer Academic/Plenum Publishers).

  • Loidl, G., Musiol, H.J., Budisa, N., Huber, R., Poirot, S., Fourmy, D. and Moroder, L. (2000). Synthesis of b-(1-azulenyl)-L-alanine as a potential blue-colored fluorescent tryptophan analog and its use in peptide synthesis. J. Pept. Sci. 6, 139–144.

    • Google Scholar
    • Export Citation
  • Mehl, R.A., Anderson, J.C., Santoro, S.W., Wang, L., Martin, A.B., King, D.S., Horn, D.M. and Schultz, P.G. (2003). Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935–939.

    • Google Scholar
    • Export Citation
  • Mendel, D., Cornish, V.W. and Schultz, P.G. (1995). Site-directed mutagenesis with an expanded genetic code. Annu. Rev. Biophys. Biomol. Struct. 24, 435–462.

    • Google Scholar
    • Export Citation
  • Minks, C. (1999). In vivo Einbau nicht-natürlicher Aminosäuren in rekombinante Proteine. PhD Thesis, Technische Universität München, München, Germany.

  • Minks, C., Huber, R., Moroder, L. and Budisa, N. (1999). Atomic mutations at the single tryptophan residue of human recombinant annexin V: effects on structure, stability, and activity. Biochemistry 38, 10649–10659.

    • Google Scholar
    • Export Citation
  • Minks, C., Alefelder, S., Moroder, L., Huber, R. and Budisa, N. (2000). Towards new protein engineering: in vivo building and folding of protein shuttles for drug delivery and targeting by the selective pressure incorporation (SPI) method. Tetrahedron 56, 9431–9442.

    • Google Scholar
    • Export Citation
  • Pratt, E.A. and Ho, C. (1974). Incorporation of fluorotryptophans into protein in Escherichia coli, and their effect on induction of b-galactosidase and lactose permease. Fed. Proc. 33, 1463–1463.

    • Google Scholar
    • Export Citation
  • Richmond, M.H. (1962). Effect of amino acid analogues on growth and protein synthesis in microorganisms. Bacteriol. Rev. 26, 398–464.

    • Google Scholar
    • Export Citation
  • Rosenthal, G. (1982). Plant nonprotein amino and imino acids. Biological, Biochemical and Toxicological Properties (New York, USA: Academic Press).

  • Ross, J.B.A., Rusinova, E., Luck, L.A. and Rousslang, K.W. (2000). Spectral enhancement of proteins by in vivo incoropration of tryptophan analogues. In: Trends in Fluorescence Spectroscopy, Vol. 6, J.R. Lakowitz, ed. (New York, USA: Plenum Publishers).

  • Ross, J.B.A., Szabo, A.G. and Hogue, C.W.V. (1997). Enhancement of protein spectra with tryptophan analogs: fluorescence spectroscopy of protein-protein and protein-nucleic acid interactions. Methods Enzymol. 278, 151–190.

    • Google Scholar
    • Export Citation
  • Santoro, S.W., Wang, L., Herberich, B., King, D.S. and Schultz, P.G. (2002). An efficient system for the evolution of aminoacyl- tRNA synthetase specificity. Nat. Biotechnol. 20, 1044–1048.

    • Google Scholar
    • Export Citation
  • Schlesinger, S. and Schlesinger, M.J. (1967). Effect of amino acid analogues on alkaline phosphatase formation in Escherichia coli K-12: substitution of triazolealanine for histidine. J. Biol. Chem. 242, 3369–3378.

    • Google Scholar
    • Export Citation
  • Seifert, M.H., Ksiazek, D., Azim, M.K., Smialowski, P., Budisa, N. and Holak, T.A. (2002). Slow exchange in the chromophore of a green fluorescent protein variant. J. Am. Chem. Soc. 124, 7932–7942.

    • Google Scholar
    • Export Citation
  • Senear, D.F., Mendelson, R.A., Stone, D.B., Luck, L.A., Rusinova, E. and Ross, J.B.A. (2002). Quantitative analysis of tryptophan analogue incorporation in recombinant proteins. Anal. Biochem. 300, 77–86.

    • Google Scholar
    • Export Citation
  • Sinha, H.K., Dogra, S.K. and Krishnamurthy, M. (1987). Excited-state and ground-state proton-transfer reactions in 5-aminoindole. Bull. Chem. Soc. Jpn. 60, 4401–4407.

    • Google Scholar
    • Export Citation
  • Steward, L.E., Collins, C.S., Gilmore, M.A., Carlson, J.E., Ross, J.B.A. and Chamberlin, A.R. (1997). In vitro site-specific incorporation of fluorescent probes into b-galactosidase. J. Am. Chem. Soc. 119, 6–11.

    • Google Scholar
    • Export Citation
  • Sykes, B.D., Weingart, H. and Schlesinger, M.J. (1974). Fluorotyrosine alkaline-phosphatase from Escherichia coli: preparation, properties, and fluorine-19 nuclear magnetic-resonance spectrum. Proc. Natl. Acad. Sci. USA 71, 469–473.

    • Google Scholar
    • Export Citation
  • Tang, Y. and Tirrell, D.A. (2001). Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial host. J. Am. Chem. Soc. 123, 11089–11090.

    • Google Scholar
    • Export Citation
  • Wang, L. and Schultz, P.G. (2002). Expanding the genetic code. Chem. Commun. 1, 1–11.

  • Wong, C.Y. and Eftink, M.R. (1998). Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137–149 fragment: spectroscopic studies. Biochemistry 37, 8938–8946.

    • Google Scholar
    • Export Citation
  • Xu, Z.J., Love, M.L., Ma, L.Y.Y., Blum, M., Bronskill, P.M., Bernstein, J., Grey, A.A., Hofmann, T., Camerman, N. and Wong, J.T.F. (1989). Tryptophanyl-tRNA synthetase from Bacillus subtilis: characterization and role of hydrophobicity in substrate recognition. J. Biol. Chem. 264, 4304–4311.

    • Google Scholar
    • Export Citation
  • Yang, F., Moss, L.G. and Phillips, G.N. (1996). The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251.

    • Google Scholar
    • Export Citation
  • Yu, Y.D., Liu, Y.Q., Shen, N., Xu, X., Xu, F., Jia, J., Jin, Y.X., Arnold, E. and Ding, J.P. (2004). Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment-insights into substrate recognition, tRNA binding, and angiogenesis activity. J. Biol. Chem. 279, 8378–8388.

    • Google Scholar
    • Export Citation
  • Zhang, Z.W., Alfonta, L., Tian, F., Bursulaya, B., Uryu, S., King, D.S. and Schultz, P.G. (2004). Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 8882–8887.

    • Google Scholar
    • Export Citation
  • Zimmer, M. (2002). Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem. Rev. 102, 759–781.

    • Google Scholar
    • Export Citation
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