Ribosome-mediated incorporation of hydrazinophenylalanine into modified peptide and protein analogues

Jennifer A. Killian, Mark D. Van Cleve, Yuda F. Shayo, Sidney Hecht

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

(S)-α-Hydrazinophenylalanyl-tRNA(Phe), an aminoacyl-tRNA derivative containing the unnatural amino acid (S)-α-hydrazinophenylalanine, was prepared in an effort to examine the stereochemical requirements of the A- site of the ribosome during in vitro protein synthesis. The (S)-α- hydrazinophenylalanine moiety was of interest because it contains two nucleophilic centers, the secondary nitrogen attached to C(α), which is normally acylated during the course of peptide bond formation, and the sterically less hindered primary nitrogen. To determine the position of acylation, (S)-α-hydrazinophenylalanyl-tRNA(Phe) was tested in an Escherichia cull in vitro protein biosynthesizing system lacking elongation factor G, such that only dipeptide products were formed. The dipeptide product mixture was analyzed by HPLC in direct comparison with authentic synthetic standards. The dipeptide assay utilizing (S)-α- hydrazinophenylalanyl-tRNA(Phe) as the A-site tRNA established that the analogue functioned well as an acceptor tRNA; HPLC analysis of the products showed that both dipeptides were formed in approximately equal amounts. When attached to a suppressor tRNA transcript, (S)-α-hydrazinophenylalanine was also incorporated into position 27 of dihydrofolate reductase in an E. coli protein synthesizing system by readthrough of a nonsense codon. This finding expands the currently accepted model of peptide bond formation at the ribosome and adds to the repertoire of peptide-like products shown to form at the peptidyltransferase center of the ribosome.

Original languageEnglish (US)
Pages (from-to)3032-3042
Number of pages11
JournalJournal of the American Chemical Society
Volume120
Issue number13
DOIs
StatePublished - Apr 8 1998
Externally publishedYes

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Dipeptides
RNA, Transfer, Phe
Transfer RNA
Ribosomes
Peptides
Proteins
Nitrogen
Peptide Elongation Factor G
High Pressure Liquid Chromatography
Peptidyl Transferases
Escherichia
Acylation
Tetrahydrofolate Dehydrogenase
Nonsense Codon
Escherichia coli Proteins
Assays
Derivatives
Amino Acids
Escherichia coli
Amino acids

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Ribosome-mediated incorporation of hydrazinophenylalanine into modified peptide and protein analogues. / Killian, Jennifer A.; Van Cleve, Mark D.; Shayo, Yuda F.; Hecht, Sidney.

In: Journal of the American Chemical Society, Vol. 120, No. 13, 08.04.1998, p. 3032-3042.

Research output: Contribution to journalArticle

Killian, Jennifer A. ; Van Cleve, Mark D. ; Shayo, Yuda F. ; Hecht, Sidney. / Ribosome-mediated incorporation of hydrazinophenylalanine into modified peptide and protein analogues. In: Journal of the American Chemical Society. 1998 ; Vol. 120, No. 13. pp. 3032-3042.
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