title: Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo.
abstract: Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: I Caglar
authors.initials: IC
authors.lastname: Tanrikulu
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Schmitt
authors.forename: Yves
authors.initials: Y
authors.lastname: Mechulam
authors.forename: William A
authors.initials: WA
authors.lastname: Goddard
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D001386:Azides / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D005434:Flow Cytometry; D006860:Hydrogen Bonding; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008718:Methionine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D009646:Norleucine / Q000031:analogs & derivatives* / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
chemical_list: D001386:Azides; C000609218:azidonorleucine; D009646:Norleucine; D008715:Methionine; D008718:Methionine-tRNA Ligase
references.citation: J Am Chem Soc. 2007 Mar 14;129(10):2744-5
references.pmid: 17305342
references.citation: Angew Chem Int Ed Engl. 2004 Dec 3;43(47):6426-63
references.pmid: 15578784
references.citation: J Am Chem Soc. 2003 Sep 17;125(37):11164-5
references.pmid: 16220915
references.citation: Angew Chem Int Ed Engl. 2002 Nov 4;41(21):4066-9
references.pmid: 12412083
references.citation: J Am Chem Soc. 2004 Nov 24;126(46):15046-7
references.pmid: 15547999
references.citation: J Am Chem Soc. 2004 Sep 1;126(34):10598-602
references.pmid: 15327317
references.citation: Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):19-24
references.pmid: 11752401
references.citation: EMBO J. 1990 May;9(5):1665-72
references.pmid: 2184035
references.citation: J Am Chem Soc. 2005 Oct 19;127(41):14150-1
references.pmid: 16218586
references.citation: J Mol Biol. 2001 Mar 2;306(4):863-76
references.pmid: 11243794
references.citation: Bioinformatics. 2005 Aug 1;21(15):3314-5
references.pmid: 15932904
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10180-10185
references.pmid: 16801548
references.citation: Angew Chem Int Ed Engl. 2006 Nov 13;45(44):7364-7
references.pmid: 17036290
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9482-7
references.pmid: 16769897
references.citation: Chembiochem. 2006 Aug;7(8):1251-7
references.pmid: 16758500
references.citation: J Am Chem Soc. 2001 Nov 7;123(44):11089-90
references.pmid: 11686725
references.citation: J Biol Chem. 1991 Sep 15;266(26):17136-41
references.pmid: 1654323
references.citation: J Am Chem Soc. 2007 Apr 25;129(16):4874-5
references.pmid: 17397163
references.citation: J Am Chem Soc. 2005 Jul 27;127(29):10136-7
references.pmid: 16028902
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12479-84
references.pmid: 15308774
references.citation: FEBS Lett. 2001 Jul 27;502(1-2):25-30
references.pmid: 11478942
references.citation: J Mol Biol. 2003 Sep 5;332(1):59-72
references.pmid: 12946347
references.citation: Nat Chem Biol. 2009 Oct;5(10):715-7
references.pmid: 19668194
references.citation: J Am Chem Soc. 2001 Nov 28;123(47):11815-6
references.pmid: 11716746
references.citation: J Mol Biol. 2003 May 16;328(5):1071-81
references.pmid: 12729742
references.citation: Methods. 2005 Jul;36(3):291-8
references.pmid: 16076455
references.citation: Mol Microbiol. 2004 May;52(3):613-9
references.pmid: 15101969
references.citation: Angew Chem Int Ed Engl. 2007;46(28):5340-3
references.pmid: 17568466
references.citation: J Mol Biol. 2001 Jun 15;309(4):925-36
references.pmid: 11399069
references.citation: J Mol Biol. 1999 Dec 17;294(5):1287-97
references.pmid: 10600385
references.citation: Bioorg Med Chem Lett. 2008 Nov 15;18(22):6004-6
references.pmid: 18845434
references.citation: Trends Biotechnol. 2006 Aug;24(8):364-71
references.pmid: 16820235
references.citation: Chembiochem. 2005 Dec;6(12):2170-3
references.pmid: 16261552
references.citation: Science. 2000 Mar 17;287(5460):2007-10
references.pmid: 10720325
references.citation: Acta Crystallogr B. 1999 Oct 1;55(Pt 5):807-809
references.pmid: 10927421