title: Site-specific incorporation of (aminooxy)acetic acid into proteins.
abstract: By employing a general biosynthetic method for the elaboration of proteins containing unnatural amino acid analogues, we incorporated (aminooxy)acetic acid into positions 10 and 27 of Escherichia coli dihydrofolate reductase. Introduction of the modified amino acid into DHFR was accomplished in an in vitro protein biosynthesizing system by readthrough of a nonsense (UAG) codon with a suppressor tRNA that had been activated with (aminooxy)acetic acid. Incorporation of the amino acid proceeded with reasonable efficiency at codon position 10 but less well at position 27. (Aminooxy)acetic acid was also incorporated into position 72 of DNA polymerase beta. Peptides containing (aminooxy)acetic acid have been shown to adopt a preferred conformation involving an eight-membered ring that resembles a gamma-turn. Accordingly, the present study may facilitate the elaboration of proteins containing conformationally biased peptidomimetic motifs at predetermined sites. The present results further extend the examples of ribosomally mediated formation of peptide bond analogues of altered connectivity and provide a conformationally biased linkage at a predetermined site. It has also been shown that the elaborated protein can be cleaved chemically at the site containing the modified amino acid.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22901, USA.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Eisenhauer
authors.forename: Sidney M
authors.initials: SM
authors.lastname: Hecht
mesh_terms: D000595:Amino Acid Sequence; D000625:Aminooxyacetic Acid / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003062:Codon; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D011506:Proteins; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D000625:Aminooxyacetic Acid; D008715:Methionine; D013762:Tetrahydrofolate Dehydrogenase; D019951:DNA Polymerase beta