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Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles

  • Anne M. Wagner
  • John B. Warner
  • Haviva E. Garrett
  • Christopher R. Walters
  • E. James PeterssonEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1337)

Abstract

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Key words

N-terminus N-end rule Aminoacyl transferase Protein modification Chemoenzymatic reaction Protein labeling Click chemistry 

Notes

Acknowledgments

This work was supported by funding from the University of Pennsylvania and the Searle Scholars Program (10-SSP-214 to EJP). HEG was supported by a summer research fellowship from Eli Lilly.

References

  1. 1.
    Varshavsky A (2011) The N-end rule pathway and regulation by proteolysis. Protein Sci 20(8):1298–1345PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134(5):866–876PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Xu GQ, Shin SBY, Jaffrey SR (2009) Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini. Proc Natl Acad Sci U S A 106(46):19310–19315PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Kleifeld O, Doucet A, Prudova A, Keller UAD, Gioia M, Kizhakkedathu JN, Overall CM (2011) Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat Protoc 6(10):1578–1611CrossRefPubMedGoogle Scholar
  5. 5.
    Taki M, Kuno A, Matoba S, Kobayashi Y, Futami J, Murakami H, Suga H, Taira K, Hasegawa T, Sisido M (2006) Leucyl/phenylalanyl-tRNA-protein transferase-mediated chemoenzymatic coupling of N-terminal arg/lys units in posttranslationally processed proteins with non-natural amino acids. Chembiochem 7(11):1676–1679CrossRefPubMedGoogle Scholar
  6. 6.
    Connor RE, Piatkov K, Varshavsky A, Tirrell DA (2008) Enzymatic N-terminal addition of noncanonical amino acids to peptides and proteins. Chembiochem 9(3):366–369CrossRefPubMedGoogle Scholar
  7. 7.
    Wagner AM, Fegley MW, Warner JB, Grindley CLJ, Marotta NP, Petersson EJ (2011) N-terminal protein modification using simple aminoacyl transferase substrates. J Am Chem Soc 133(38):15139–15147PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Ninnis RL, Spall SK, Talbo GH, Truscott KN, Dougan DA (2009) Modification of PATase by L/F-transferase generates a ClpS-dependent N-end rule substrate in Escherichia coli. EMBO J 28(12):1732–1744PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Leibowitz MJ, Soffer RL (1969) A soluble enzyme from Escherichia coli which catalyzes transfer of leucine and phenylalanine from tRNA to acceptor proteins. Biochem Biophys Res Commun 36(1):47–53CrossRefPubMedGoogle Scholar
  10. 10.
    Scarpulla RC, Deutch CE, Soffer RL (1976) Transfer of methionyl residues by leucyl, phenylalanyl-transfer-RNA-protein transferase. Biochem Biophys Res Commun 71(2):584–589CrossRefPubMedGoogle Scholar
  11. 11.
    Kwon YT, Reiss Y, Fried VA, Hershko A, Yoon JK, Gonda DK, Sangan P, Copeland NG, Jenkins NA, Varshavsky A (1998) The mouse and human genes encoding the recognition component of the N-end rule pathway. Proc Natl Acad Sci U S A 95(14):7898–7903PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Kwon YT, Kashina AS, Davydov IV, Hu RG, An JY, Seo JW, Du F, Varshavsky A (2002) An essential role of N-terminal arginylation in cardiovascular development. Science 297(5578):96–99CrossRefPubMedGoogle Scholar
  13. 13.
    Ferber S, Ciechanover A (1987) Role of arginine-transfer RNA in protein-degradation by the ubiquitin pathway. Nature 326(6115):808–811CrossRefPubMedGoogle Scholar
  14. 14.
    Kwon YT, Xia ZX, Davydov IV, Lecker SH, Varshavsky A (2001) Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3 alpha) of the N-end rule pathway. Mol Cell Biol 21(23):8007–8021PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Wang JL, Han XM, Saha S, Xu T, Rai R, Zhang FL, Wolf YI, Wolfson A, Yates JR, Kashina A (2011) Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo. Chem Biol 18(1):121–130PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Tanaka T, Wagner AM, Warner JB, Wang YJ, Petersson EJ (2013) Expressed protein ligation at methionine: N-terminal attachment of homocysteine, ligation, and masking. Angew Chem Int Ed Engl 52(24):6210–6213CrossRefPubMedGoogle Scholar
  17. 17.
    Sletten EM, Bertozzi CR (2009) Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed 48(38):6974–6998CrossRefGoogle Scholar
  18. 18.
    Debets MF, van Berkel SS, Schoffelen S, Rutjes F, van Hest JCM, van Delft FL (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition. Chem Commun 46(1):97–99CrossRefGoogle Scholar
  19. 19.
    Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (2002) A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed 41(14):2596–2599CrossRefGoogle Scholar
  20. 20.
    Soffer RL (1973) Peptide acceptors in leucine, phenylalanine transfer-reaction. J Biol Chem 248(24):8424–8428PubMedGoogle Scholar
  21. 21.
    Link AJ, Vink MKS, Agard NJ, Prescher JA, Bertozzi CR, Tirrell DA (2006) Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc Natl Acad Sci U S A 103(27):10180–10185PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Taiji M, Yokoyama S, Miyazawa T (1983) Transacylation rates of (aminoacyl)adenosine moiety at the 3′-terminus of aminoacyl transfer ribonucleic acid. Biochemistry 22:3220–3225CrossRefPubMedGoogle Scholar
  23. 23.
    Watanabe K, Toh Y, Suto K, Shimizu Y, Oka N, Wada T, Tomita K (2007) Protein-based peptide-bond formation by aminoacyl-tRNA protein transferase. Nature 449(7164):867–871CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anne M. Wagner
    • 1
  • John B. Warner
    • 1
  • Haviva E. Garrett
    • 1
  • Christopher R. Walters
    • 1
  • E. James Petersson
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of PennsylvaniaPhiladelphiaUSA

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