PROJECT SUMMARY / ABSTRACT The long-term goal of the application is to understand why and how bacterial lipoproteins in the medically important Firmicutes phylum are structurally modified. Lipoproteins are ubiquitous membrane associated proteins tethered to the bacterial surface through a conserved acylated cysteine residue located at the N-terminus. However, lipoprotein acylation states can vary in chain number, length, and attachment position depending on the bacterial source. Acylation states are also dynamically regulated in the same strain by particular growth environments, including by excess copper which induces expression of select N-terminal modifying genes. This project aims to utilize ultra high-pressure liquid chromatography (uHPLC) to characterize reconstituted lipoprotein N-terminal modification systems in vitro. Assays will help determine enzyme mechanisms, identify acyl chain donors, and to test important active site residues. The second part of the project will utilize HPLC to isolate and purify various model lipopeptides representing each of the known N-terminal chemotype classes (free a-amino, N-acyl, N-acetyl, and lyso-form). The chemotype panel will then be analyzed for copper binding and susceptibility to oxidation. The project will thus advance our understanding of acyl transfer biochemistry and provide insight on the physiological function of lipoprotein modifications.