Elongation Factor P Modulates the Incorporation of Structurally Diverse Noncanonical Amino Acids into Escherichia coli Dihydrofolate Reductase

J Am Chem Soc. 2023 Nov 1;145(43):23600-23608. doi: 10.1021/jacs.3c07524. Epub 2023 Oct 23.

Abstract

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acids* / chemistry
  • Escherichia coli* / metabolism
  • Peptide Elongation Factors / metabolism
  • Peptides / chemistry
  • Proline / metabolism
  • Tetrahydrofolate Dehydrogenase / metabolism

Substances

  • factor EF-P
  • Amino Acids
  • Tetrahydrofolate Dehydrogenase
  • Peptide Elongation Factors
  • Peptides
  • Proline