Formation of Ubiquitin Dimers via Azide–Alkyne Click Reaction

  • Silvia Eger
  • Martin Scheffner
  • Andreas Marx
  • Marina RubiniEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 832)


The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.

Key words

Ubiquitin chains Artificial amino acids Azidohomoalanine (Aha) Pyrrolysine analogue (Plk) Click reaction Cu(I)-catalyzed Huisgen azide–alkyne cycloaddition Triazole linkage 



We gratefully acknowledge funding by the Boehringer Ingelheim Fonds (S.E.), the EU-network of excellence RUBICON (M.S.) and the DFG (M.S., A.M.).


  1. 1.
    Hershko A, Ciechanover A (1998) The ubiquitin system. Annu. Rev. Biochem. 67:425–479.PubMedCrossRefGoogle Scholar
  2. 2.
    Hicke L, Dunn R (2003) Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell. Dev. Biol. 19:141–172.PubMedCrossRefGoogle Scholar
  3. 3.
    Pickart CM (2001) Mechanisms underlying ubiquitylation. Annu. Rev. Biochem. 70:503–533.PubMedCrossRefGoogle Scholar
  4. 4.
    Kerscher O, Felberbaum R, Hochstrasser M (2006) Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell. Dev. Biol. 222:159–180.CrossRefGoogle Scholar
  5. 5.
    Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 21:921–926.PubMedCrossRefGoogle Scholar
  6. 6.
    Li W, Ye Y (2008) Polyubiquitin chains: function, structures and mechanisms, Birkäuser Verlag, Basel.Google Scholar
  7. 7.
    Hershko A, Ciechanover A (1992) The ubiquitin system for protein degradation. Annu. Rev. Biochem. 61:761–807.PubMedCrossRefGoogle Scholar
  8. 8.
    Williamson A, Wickliffe KE, Mellone BG et al (2009) Identification of a physiological E2 module for the human anaphase-promoting complex. PNAS 106:18213–18218.PubMedCrossRefGoogle Scholar
  9. 9.
    Al-Hakim AK, Zagorska A, Chapman L et al (2008) Control of AMPK-related kinases by USP9X and atypical Lys29/Lys33-linked polyubiquitin chains. Journal of Biochemistry 411:249–260.CrossRefGoogle Scholar
  10. 10.
    Budisa N (2004) Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. Angewandte Chemie International Edition 43:6426–6463.CrossRefGoogle Scholar
  11. 11.
    Young TS, Schultz PG (2010) Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon. J. Biol. Chem. 285:11039–11044.PubMedCrossRefGoogle Scholar
  12. 12.
    Kiick KL, Saxon E, Tirrell DA et al (2002) Incorporation of azides into revombinant proteins for chemosolective modification by the Staudinger ligation. PNAS 99:19–24.PubMedCrossRefGoogle Scholar
  13. 13.
    Kasteren SIv, Kramer HB, Jensen HH et al (2007) Expanding the diversity of chemical protein modification allows post-translational mimicry. Nature 446:1105–1109.Google Scholar
  14. 14.
    Shanmugham A, Fish A, Luna-Vargas MPA et al (2010) Nonhydrolyzable ubiquitin-isopeptide isosteres as deubiquitinating enzyme probes. J. Am. Chem. Soc. 132:8834–8835.PubMedCrossRefGoogle Scholar
  15. 15.
    Kaya E, Gutsmiedl K, Vrabel M et al (2009) Synthesis of Threefold Glycosylated Proteins using Click Chemistry and Genetically Encoded Unnatural Amino Acids. ChemBioChem 10:2858–2861.PubMedCrossRefGoogle Scholar
  16. 16.
    Nguyen DP, Lusic H, Neumann H et al (2009) Genetic Encoding and Labeling of Aliphatic Azides and Alkynes in Recombinant Proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA Pair and Click Chemistry. Journal of the American Chemical Society 131:8720–8721.PubMedCrossRefGoogle Scholar
  17. 17.
    Fekner T, Li X, Lee MM et al (2009) A Pyrrolysine Analogue for Protein Click Chemistry. Angew. Chem. Int. Ed. 48:1633 –1635.CrossRefGoogle Scholar
  18. 18.
    Masson J-M, Miller JH (1986) Expression of synthetic suppressor tRNA genes under the control of a synthetic promoter. Gene 47:179–183.PubMedCrossRefGoogle Scholar
  19. 19.
    Young RA (1979) Transcription Termination in the Escherichia coli Ribosomal RNA Operon rrnC. THE JOURNAL OF BIOLOGICAL CHEMISTRY 254:12725–12731.PubMedGoogle Scholar
  20. 20.
    Huisgen R (1963) 1,3-Dipolar Cycloadditions. Past and Future. Angew. Chem. Int. Ed. 2:565–598.CrossRefGoogle Scholar
  21. 21.
    Rostovtsev VV, Green LG, Fokin VV et al (2002) A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. 41:2596–2599.CrossRefGoogle Scholar
  22. 22.
    Tornoe CW, Christensen C, Meldal M (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67:3057–3064.PubMedCrossRefGoogle Scholar
  23. 23.
    Link AJ, Vink MKS, Tirrell DA (2007) Preparation of the functionoalized methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer. Nature Protocols 2:1879–1883.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Silvia Eger
    • 1
  • Martin Scheffner
    • 2
  • Andreas Marx
    • 1
  • Marina Rubini
    • 1
    Email author
  1. 1.Department of Chemistry, Konstanz Research School Chemical BiologyUniversity of KonstanzKonstanzGermany
  2. 2.Department of Biology, Konstanz Research School Chemical BiologyUniversity of KonstanzKonstanzGermany

Personalised recommendations