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

  • Nediljko Budisa and Prajna Paramita Pal
From the journal Biological Chemistry


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, 925–936.10.1006/jmbi.2001.4699Search in Google Scholar

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.Search in Google Scholar

Brawerman, G. and Ycas, M. (1957). Incorporation of the amino acid analog tryptazan into the protein of Escherichia coli. Arch. Biochem. Biophys.68, 112–117.10.1016/0003-9861(57)90331-4Search in Google Scholar

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, 305–308.10.1042/bj2490305Search in Google Scholar

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. 89–98.10.1007/978-3-642-59337-6_11Search in Google Scholar

Budisa, N. (2004a). Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angew. Chem. 45, in press.10.1002/chin.200512291Search in Google Scholar

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

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. USA95, 455–459.10.1073/pnas.95.2.455Search in Google Scholar

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, 41–51.10.1096/fasebj.13.1.41Search in Google Scholar

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, 1626–1635.10.1007/s000180050401Search in Google Scholar

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, 1281–1292.10.1110/ps.51601Search in Google Scholar

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, 4066–4069.10.1002/1521-3773(20021104)41:21<4066::AID-ANIE4066>3.0.CO;2-6Search in Google Scholar

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, 191–202.10.1515/BC.2004.038Search in Google Scholar

Burley, S.K. and Petsko, G.A. (1988). Weakly polar interactions in proteins. Adv. Protein Chem.39, 125–189.10.1016/S0065-3233(08)60376-9Search in Google Scholar

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. USA48, 1086–1098.10.1073/pnas.48.6.1086Search in Google Scholar

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

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

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, 1489–1502.10.1110/ps.03542104Search in Google Scholar

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. Macromolecules29, 1442–1444.10.1021/ma9510698Search in Google Scholar

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. Structure3, 17–31.10.1016/S0969-2126(01)00132-0Search in Google Scholar

Dougherty, D.A. (1996). Cation-p interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science271, 163–168.10.1126/science.271.5246.163Search in Google Scholar

Evans, C.S. and Bell, E.A. (1980). Neuroactive plant amino acids and amines. Trends Neurosci.3, 70–72.10.1016/0166-2236(80)90027-2Search in Google Scholar

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, 1505–1514.10.1110/ps.8.7.1505Search in Google Scholar PubMed PubMed Central

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, 29188–29194.10.1074/jbc.M102815200Search in Google Scholar PubMed

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, 40324–40328.10.1074/jbc.M003696200Search in Google Scholar

Hohsaka, T. and Sisido, M. (2002). Incorporation of non-natural amino acids into proteins. Curr. Opin. Chem. Biol.6, 809–815.10.1016/S1367-5931(02)00376-9Search in Google Scholar

Hortin, G. and Boime, I. (1983). Applications of amino acid analogs for studying co-translational and posttranslational modifications of proteins. Methods Enzymol.96, 777–784.10.1016/S0076-6879(83)96065-2Search in Google Scholar

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, 2148–2151.10.1002/1521-3773(20000616)39:12<2148::AID-ANIE2148>3.0.CO;2-7Search in Google Scholar

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, 1267–1271.10.1246/bcsj.50.1267Search in Google Scholar

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

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, 139–144.10.1002/(SICI)1099-1387(200003)6:3<139::AID-PSC240>3.0.CO;2-6Search in Google Scholar

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, 935–939.10.1021/ja0284153Search in Google Scholar

Mendel, D., Cornish, V.W. and Schultz, P.G. (1995). Site-directed mutagenesis with an expanded genetic code. Annu. Rev. Biophys. Biomol. Struct.24, 435–462.10.1146/ in Google Scholar

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

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. Biochemistry38, 10649–10659.10.1021/bi990580gSearch in Google Scholar

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. Tetrahedron56, 9431–9442.10.1016/S0040-4020(00)00827-9Search in Google Scholar

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.Search in Google Scholar

Richmond, M.H. (1962). Effect of amino acid analogues on growth and protein synthesis in microorganisms. Bacteriol. Rev.26, 398–464.10.1128/br.26.4.398-420.1962Search in Google Scholar

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

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).Search in Google Scholar

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, 151–190.10.1016/S0076-6879(97)78010-8Search in Google Scholar

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, 1044–1048.10.1038/nbt742Search in Google Scholar

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, 3369–3378.10.1016/S0021-9258(18)95919-3Search in Google Scholar

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, 7932–7942.10.1021/ja0257725Search in Google Scholar PubMed

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, 77–86.10.1006/abio.2001.5441Search in Google Scholar PubMed

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, 4401–4407.10.1246/bcsj.60.4401Search in Google Scholar

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, 6–11.10.1021/ja963023fSearch in Google Scholar

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. USA71, 469–473.10.1073/pnas.71.2.469Search in Google Scholar PubMed PubMed Central

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, 11089–11090.10.1021/ja016652kSearch in Google Scholar PubMed

Wang, L. and Schultz, P.G. (2002). Expanding the genetic code. Chem. Commun.1, 1–11.10.1039/b108185nSearch in Google Scholar

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. Biochemistry37, 8938–8946.10.1021/bi971862oSearch in Google Scholar

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, 4304–4311.10.1016/S0021-9258(18)83740-1Search in Google Scholar

Yang, F., Moss, L.G. and Phillips, G.N. (1996). The molecular structure of green fluorescent protein. Nat. Biotechnol.14, 1246–1251.10.1038/nbt1096-1246Search in Google Scholar PubMed

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, 8378–8388.10.1074/jbc.M311284200Search in Google Scholar PubMed

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. USA101, 8882–8887.10.1073/pnas.0307029101Search in Google Scholar PubMed PubMed Central

Zimmer, M. (2002). Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem. Rev.102, 759–781.10.1021/cr010142rSearch in Google Scholar PubMed

Published Online: 2005-06-01
Published in Print: 2004-10-01

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