Skip to main content
SpringerLink
Log in

In vivo engineering of proteins with nitrogen-containing tryptophan analogs

  • Mini-Review
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Recently, it has become possible to reprogram the protein synthesis machinery such that numerous noncanonical amino acids can be translated into target sequences yielding tailor-made proteins. The canonical amino acid tryptophan (Trp) encoded by a single nucleotide triplet (UGG) is a particularly interesting target for protein engineering and design. Trp-residues can be substituted with a variety of analogs and surrogates generated biosynthetically or by organic chemistry. Among them, nitrogen-containing tryptophan analogs occupy a central position, as they have distinct chemical properties in comparison with aliphatic amines and imines. They resemble purine bases of DNA and share their capacity for pH-sensitive intramolecular charge transfer. These special properties of the analogs can be directly transmitted into related protein structures via in vivo ribosome-mediated translation. Proteins expressed in this way are further endowed with unique properties like new spectral, altered redox and titration features or might serve as useful biomaterials. We present and discuss current works and future developments in protein engineering with nitrogen-containing tryptophan analogs and related compounds as well as their relevance for academic and applicative research.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  • Ardell DH, Sella G (2001) On the evolution of redundancy in genetic codes. J Mol Evol 53:269–281

    Article  CAS  PubMed  Google Scholar 

  • 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 309:925–936

    Article  CAS  PubMed  Google Scholar 

  • 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 328:1071–1081

    Article  CAS  PubMed  Google Scholar 

  • Baldwin RL (2002) Protein folding—making a network of hydrophobic clusters. Science 295:1657–1658

    Article  CAS  PubMed  Google Scholar 

  • Bergstrom DE (2004) Orthogonal base pairs continue to evolve. Chem Biol 11:18–20

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Budisa N (2004) Prolegomena to future efforts on genetic code engineering by expanding its amino acid repertoire. Angew Chem Angew Chem Int Ed 43:3387–3428

    Google Scholar 

  • Budisa N (2005a) Amino acids and codons—code organisation and protein structure. In: Engineering the genetic code. Wiley, Weinheim, pp 66–90

    Google Scholar 

  • Budisa N (2005b) A brief history of an expanded amino acid repertoire. In: Engineering the genetic code. Wiley, Weinheim, pp 13–28

    Google Scholar 

  • Budisa N (2005c) Reprogramming the cellular translation machinery. In: Engineering the genetic code. Wiley, Weinheim, pp 90–184

    Book  Google Scholar 

  • Budisa N (2005d) Some practical potentials of reprogrammed cellular translation. In: Engineering the genetic code. Wiley, Weinheim, pp 213–261

    Google Scholar 

  • Budisa N, Pal PP (2004) Designing novel spectral classes of proteins with tryptophan-expanded genetic code. Biol Chem 385:893–904

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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 10:1281–1292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Budisa N, Pal PP, Alefelder S, Birle P, Krywcun T, Rubini M, Wenger W, Bae JH, Steiner T (2004a) Probing the role of tryptophans in Aequorea victoria green fluorescent proteins with an expanded genetic code. Biol Chem 385:191–202

    CAS  PubMed  Google Scholar 

  • Budisa N, Pipitone O, Slivanowiz I, Rubini M, Pal PP, Holak TA, Huber R, Gelmi ML (2004) Efforts toward the design of ‘Teflon’ proteins: in vivo translation with trifluorinated leucine and methionine analogues. Chem Biodiver 1:1465–1475

    Article  CAS  Google Scholar 

  • Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird DA, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carter CW (2004) Tryptophanyl-tRNA Synthetase. In: Ibba M, Francklyn C, Cusack S (eds) Aminoacyl-tRNA synthetases. Landes Bioscience, Austin

    Google Scholar 

  • Chapeville F, Ehrenstein GV, Benzer S, Weisblum B, Ray WJ, Lipmann F (1962) On role of soluble ribonucleic acid in coding for amino acids. Proc Natl Acad Sci USA 48:1086–1092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cohen GN, Munier R (1959) Effects of structural analogues of amino acids on growth and synthesis of proteins and enzymes in Escherichia coli. Biochim Biophys Acta 31:347–356

    Article  CAS  PubMed  Google Scholar 

  • Crick FHC (1968) Origin of genetic code. J Mol Biol 38:367–379

    Article  CAS  PubMed  Google Scholar 

  • Doublie S, Bricogne G, Gilmore C, Carter CW (1995) Tryptophanyl-transfer-Rna synthetase crystal-structure reveals an unexpected homology to tyrosyl-transfer-Rna synthetase. Structure 3:17–31

    Article  CAS  PubMed  Google Scholar 

  • Dougherty DA (1996) Cation—interactions in chemistry and biology: a new view of Benzene, Phe, Tyr, and Trp. Science 271:163–168

    Article  CAS  PubMed  Google Scholar 

  • Dryden DFT, Tock MR (2006) DNA mimicry by proteins. Biochem Soc Trans 34(Part 2):317–319

    Article  CAS  PubMed  Google Scholar 

  • Evans CS, Bell EA (1980) Neuroactive plant amino acids and amines. Trends Neurosci 3:70–72

    Article  CAS  Google Scholar 

  • Ezekiel DH (1965) False feedback inhibition of aromatic amino acid biosynthesis by beta-2-thienylalanine. Biochim Biophys Acta 95:54–62

    Article  CAS  PubMed  Google Scholar 

  • Hendrickson TL, de Crecy-Lagard V, Schimmel P (2004) Incorporation of nonnatural amino acids into proteins. Ann Rev Biochem 73:147–176

    Article  CAS  PubMed  Google Scholar 

  • Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T, Nakayama H, Takio K, Yabuki T, Kigawa T, Kodama K, Nishikawa K, Yokoyama S (2002) An unnatural base pair for incorporating amino acid analogs into proteins. Nat Biotechnol 20:177–182

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Hohsaka T, Sato K, Sisido M, Takai K, Yokoyama S (1993) Adaptability of nonnatural aromatic amino acids to the active center of the Escherichia coli ribosomal A-site. FEBS Lett 335:47–50

    Article  CAS  PubMed  Google Scholar 

  • 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 18:273–278

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Koopman G, Reutelingsperger C, Kuijten G, Keehnen R, Pals S, van Oers M (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415–1420

    Article  CAS  PubMed  Google Scholar 

  • Labas YA, Gurskaya NG, Yanushevich YG, Fradkov AF, Lukyanov KA, Lukyanov SA, Matz MV (2002) Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci USA 99:4256–4261

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lakowitz JR (1999) Protein fluorescence. In: Principles of fluorescence spectroscopy. Kluwer/Plenum, New York, pp 445–486

    Book  Google Scholar 

  • Levitt M, Gerstein M, Huang E, Subbiah S, Tsai J (1997) Protein folding: the endgame. Ann Rev Biochem 66:549–579

    Article  CAS  PubMed  Google Scholar 

  • Martynov VI, Savitsky AP, Martynova NY, Savitsky PA, Lukyanov KA, Lukyanov SA (2001) Alternative cyclization in GFP-like proteins family. J Biol Chem 276:21012–21016

    Article  CAS  PubMed  Google Scholar 

  • Minks C, Alefelder S, Moroder L, Huber R, 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

    Article  CAS  Google Scholar 

  • Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395

    Article  CAS  PubMed  Google Scholar 

  • Palm GJ, Wlodawer A (1999) Spectral variants of green fluorescent protein. In: Green fluorescent protein, vol. 302, pp 378–394

  • Pardee AB, Shore VG, Prestidge LS (1956) Incorporation of azatryptophan into proteins of bacteria and bacteriophage. Biochim Biophys Acta 21:406–407

    Article  CAS  PubMed  Google Scholar 

  • Rosenthal G (1982) Plant nonprotein amino and imino acids. Biological, biochemical and toxicological properties. Academic, New York

    Google Scholar 

  • Ross JBA, Szabo AG, Hogue CWV (1997) Enhancement of protein spectra with tryptophan analogs: fluorescence spectroscopy of protein–protein and protein–nucleic acid interactions. In: Fluorescence spectroscopy, vol 278, pp 151–190

  • Ross JBA, Rusinova E, Luck LA, Rousslang KW (2000) Spectral enhancement of proteins by in vivo incorporation of tryptophan analogues. In: Lakowitz JR (ed) Trends in fluorescence spectroscopy, vol 6. Plenum, New York

    Google Scholar 

  • Rubini M, Lepthien S, Golbik R, Budisa N (2006) Aminotryptophan-containing barstar: structure–function tradeoff in protein design and engineering with an expanded genetic code. Proteomics 1764:1147–1158

    CAS  Google Scholar 

  • Schlesinger S, Schlesinger MJ (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

    CAS  PubMed  Google Scholar 

  • Shafirovich V, Geacintov NE (2005) Spectroscopic investigations of charge transfer in DNA. In: Wagenknecht HA (ed) Charge transfer in DNA, vol. Wiley, Weinheim, pp 175–193

    Google Scholar 

  • Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  CAS  PubMed  Google Scholar 

  • Shimomura O (1979) Structure of the chromophore of aequorea green fluorescent protein. FEBS Lett 104:220–222

    Article  CAS  Google Scholar 

  • Sinha HK, Dogra SK, Krishnamurthy M (1987) Excited-state and ground-state proton-transfer reactions in 5-aminoindole. Bull Chem Soc Jpn 60:4401–4407

    Article  CAS  Google Scholar 

  • Sonneborn TM (1965) Degeneracy of the genetic code: extent, nature and genetic implications. In: Bryson V, Vogel HJ (eds) Evolving genes and proteins. Academic, New York, pp 377–397

    Chapter  Google Scholar 

  • Sridevi K, Juneja J, Bhuyan AK, Krishnamoorthy G, Udgaonkar JB (2000) The slow folding reaction of barstar: the core tryptophan region attains tight packing before substantial secondary and tertiary structure formation and final compaction of the polypeptide chain. J Mol Biol 302:479–495

    Article  CAS  PubMed  Google Scholar 

  • Swanson R, Hoben P, Sumner-Smith M, Uemura H, Watson L, Soll D (1998) Accuracy of in vivo aminoacylation requires proper balance of tRNA and aminoacyl-tRNA synthetase. Science 242:1548–1551

    Article  Google Scholar 

  • Sykes BD, Weingart H, Schlesin M (1974) Fluorotyrosine alkaline-phosphatase from Escherichia coli—preparation, properties, and fluorine-19 nuclear magnetic-resonance spectrum. Proc Natl Acad Sci USA 71:469–473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Szathmary E (2003) Why are there four letters in the genetic alphabet? Nat Rev Genet 4:995–1001

    Article  CAS  PubMed  Google Scholar 

  • Tang Y, Tirrell DA (2001) Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial host. J Am Chem Soc 123:11089–11090

    Article  CAS  PubMed  Google Scholar 

  • Tidor B, Lee LP (2001) Barstar is electrostatically optimized for tight binding to barnase. Nat Struct Biol 8:73–76

    Article  PubMed  CAS  Google Scholar 

  • Trinquier G, Sanejouand YH (1998) Which effective property of amino acids is best preserved by the genetic code? Protein Eng 11:153–169

    Article  CAS  PubMed  Google Scholar 

  • Turesky RJ (2004) The role of genetic polymorphisms in metabolism of carcinogenic heterocyclic aromatic amines. Curr Drug Metab 5:169–180

    Article  CAS  PubMed  Google Scholar 

  • Twine SM, Szabo AG (2003) Fluorescent amino acid analogs. Methods Enzymol 360:104–127

    Article  CAS  PubMed  Google Scholar 

  • Vaughan M, Steinberg D (1959) The specificty of protein synthesis. In: Anfinsen CB, Anson ML, Bailey K, Edsall JT (eds) Advances in protein chemistry, vol XIV. Academic, New York and London, pp 116–173

    Google Scholar 

  • Verkhusha VV, Lukyanov KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22:289–296

    Article  CAS  PubMed  Google Scholar 

  • 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 6:1267–1277

    Article  CAS  PubMed  Google Scholar 

  • Walsh CT (2006) Posttranslational modifications of proteins: expanding nature’s inventory. Roberts, Englewood, Colorado

    Google Scholar 

  • Weber AL, Miller SL (1981) Reasons for the occurrence of the 20 coded protein amino acids. J Mol Evol 17:273–284

    Article  CAS  PubMed  Google Scholar 

  • Woese CR, Dugre DH, Dugre SA, Kondo M, Saxinger WC (1966) On fundamental nature and evolution of genetic code. Cold Spring Harbor Symp Quant Biol 31:723–736

    Article  CAS  PubMed  Google Scholar 

  • Wolfenden R, Radzicka A (1986) How hydrophilic is tryptophan. Trends Biochem Sci 11:69–70

    Article  CAS  Google Scholar 

  • Wong TTF (1998) Evolution of the genetic code. Microbiol Sci 5:174–181

    Google Scholar 

  • Xu ZJ, Love ML, Ma LYY, Blum M, Bronskill PM, Bernstein J, Grey AA, Hofmann T, Camerman N, Wong JTF (1989) Tryptophanyl-tRNA synthetase from Bacillus subtilis: characterization and role of hydrophobicity in substrate recognition. J Biol Chem 264:4304–4311

    CAS  PubMed  Google Scholar 

  • Zhang ZW, Alfonta L, Tian F, Bursulaya B, Uryu S, King DS, Schultz PG (2004) Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells. Proc Natl Acad Sci USA 101:8882–8887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Biotechnology, Max-Planck-Institut für Biochemie, BioFuture Independent Research Group, Am Klopferspitz 18, 82152, Martinsried, Germany

    Sandra Lepthien, Birgit Wiltschi, Bojana Bolic & Nediljko Budisa

Authors
  1. Sandra Lepthien
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Birgit Wiltschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bojana Bolic
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nediljko Budisa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nediljko Budisa.

Additional information

The term noncanonical amino acid refers to an amino acid that does not belong, in contrast to a canonical amino acid, to the genetically encoded, proteinogenic amino acids. The term analog defines a strict isosteric exchange of a canonical/noncanonical amino acid (e.g., tryptophan/azatryptophan), while the term surrogate defines a nonisosteric change (e.g., tryptophan/azulene). Mutant denotes a protein in which the wild-type sequence was changed by site-directed mutagenesis (codon manipulation on the DNA level) within the repertoire of the standard amino acids. Variant denotes a protein in which one or more canonical amino acids derived from a wild-type or a mutant sequence were replaced by a noncanonical one (expanded amino acid repertoire, codon reassignment on the protein translation level).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lepthien, S., Wiltschi, B., Bolic, B. et al. In vivo engineering of proteins with nitrogen-containing tryptophan analogs. Appl Microbiol Biotechnol 73, 740–754 (2006). https://doi.org/10.1007/s00253-006-0665-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0665-2

Keywords

Search

Navigation