Expression and In Vivo Characterization of the Antimicrobial Peptide Oncocin and Variants Binding to Ribosomes.
2020
Peptides have important biomedical
applications, but poor correlation
between in vitro and in vivo activities
can limit their development for clinical use. The ability to generate
peptides and monitor their expression with new mass spectrometric
methods and biological activities in vivo would be
an advantage for the discovery and improvement of peptide-based drugs.
In this study, a plasmid-based system was used to express the ribosome-targeting
peptide oncocin (19 amino acids, VDKPPYLPRPRPPRRIYNR)
and to determine its direct antibacterial effects on Escherichia
coli. Previous biochemical and structure studies showed that
oncocin targets the bacterial ribosome. The oncocin peptide generated in vivo strongly inhibits bacterial growth. In vivo dimethyl sulfate footprinting of oncocin on the rRNA gives results
that are consistent with those of in vitro studies
but reveals additional binding interactions with E. coli ribosomes. Furthermore, expression of truncated or mutated peptides
reveals which amino acids are important for antimicrobial activity.
Overall, the in vivo peptide expression system can
be used to investigate biological activities and interactions of peptides
with their targets within the cellular environment and to separate
contributions of the sequence to cellular transport. This strategy
has future applications for improving the effectiveness of existing
peptides and developing new peptide-based drugs.
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