Work

Systematic Profiling of Peptide Substrate Specificity in N-Terminal Processing by Methionine Aminopeptidase Using mRNA Display and an Unnatural Methionine Analogue

Year: 2026

Type: article

Abstract: Methionine aminopeptidase (MAP) is useful in chemical biology research for N-terminal processing of peptides and proteins and in medicine as a potential therapeutic target. These technologies can benefit from a precise understanding of the enzyme's substrate specificity profiled over a wide chemical space, including not just natural substrates, peptides containing N-terminal Met, but also unnatural peptide substrates containing N-terminal Met analogues that are also cleaved by MAP like homopropargylglycine (HPG) and azidohomoalanine (AHA). A few studies have profiled substrate specificity for cleavage of N-terminal Met, but none have systematically done so using N-terminal Met analogues. Therefore, we devised a high-throughput profiling experiment based on mRNA display and NGS to probe MAP's substrate specificity using N-terminal HPG. From subgroup analysis of either single residues or two-residue combinations, we could establish the impact of residue identity at various positions downstream from the cleavage site. To validate the selection results, a collection of short peptides was chemically synthesized and assayed for cleavage efficiency, where we observed reasonable agreement with selection data. Results generally followed previously reported trends using N-terminal Met, the strongest trend being that the second residue (P1' position) had the greatest impact on MAP cleavage efficiency with moderate impacts discerned for residues further downstream which could be rationalized through modeling the enzyme-substrate interaction.

Source: ACS Chemical Biology

DOI: 10.1021/acschembio.5c00680