%0 Journal Article
%T Enhancement of N-Methyl Amino Acid Incorporation into Proteins and Peptides Using Modified Bacterial Ribosomes and Elongation Factor P.
%A Zhang C
%A Chen S
%A Fu X
%A Dedkova LM
%A Hecht SM
%J ACS Chem Biol
%V 19
%N 6
%D 2024 Jun 21
%M 38769080
%F 4.634
%R 10.1021/acschembio.4c00165
%X N-Methylated amino acids are constituents of natural bioactive peptides and proteins. Nα-methylated amino acids appear abundantly in natural cyclic peptides, likely due to their constraint of peptide conformation and contribution to peptide stability. Peptides containing Nα-methylated amino acids have long been prepared by chemical synthesis. While such natural peptides are not produced ribosomally, recent ribosomal strategies have afforded Nα-methylated peptides. Presently, we define new strategies for the ribosomal incorporation of Nα-methylated amino acids into peptides and proteins. First, we identify modified ribosomes capable of facilitating the incorporation of six N-methylated amino acids into antibacterial scorpion peptide IsCT. Also synthesized analogously was a protein domain (RRM1) from hnRNP LL; improved yields were observed for nearly all tested N-methylated amino acids. Computational modeling of the ribosomal assembly illustrated how the distortion imposed by N-methylation could be compensated by altering the nucleotides in key 23S rRNA positions. Finally, it is known that incorporation of multiple prolines (an N-alkylated amino acid) ribosomally can be facilitated by bacterial elongation factor P. We report that supplementing endogenous EF-P during IsCT peptide and RRM1 protein synthesis gave improved yields for most of the N-methylated amino acids studied.