Browsing by Subject "Peptides"

Natural products belonging to the lipid II-binding family act as potent antimicrobial agents by disrupting cell wall biosynthesis via sequestering the late-stage intermediate lipid II. However, the emergence of resistance mechanisms and poor bioavailability have hindered the utility of these molecules as promising therapeutic intervention strategies to combat pathogenic bacterial infections. Gaining a deeper understanding of structural components and biosynthetic pathways can lead to the creation of second-generation derivatives to improve bioactivity and pharmacological properties. To explore this superfamily, we have used bioanalytical, biochemical, synthetic, computational, and enzymatic approaches that have been applied to three distinct projects. The first includes efforts to characterize the relationship between structural feature and bioactivity for the lipid II-binding CDA (calcium dependent antibiotic), malacidin. Through a series of minimally complex analogs, we determined non-proteinogenic amino acids and the N-acyl fatty acid moiety are essential for bioactivity. For the second project, we investigated a conserved mechanism of action for phylogenetically-related natural products within the lasso peptide subfamily. This work led to the discovery of a novel class I lasso peptide, arcumycin, and we validated a conserved mechanism of action for Actinobacteria-produced lasso peptides in targeting lipid II biosynthesis. Our last project sought to elucidate the mechanism of lipoinitiation for the ramoplanin family of molecules. Through a series of bioactivity assays, we found the transfer to the acyl carrier protein (ACP) in a fatty acyl-AMP ligase (FAAL)-dependent manner determined the specificity of lipids selected in the biosynthetic process. Collectively, through each project we have gained a deeper understanding of the structural elements and biosynthetic pathways of lipid II-binding antimicrobials.

Copper was first released into the environment as the result of the mass generation of oxygen from photosynthetic bacteria roughly 2.7 billion years ago. While it proved to be poisonous to early life on Earth, those that met the evolutionary challenge utilized the metal as a cofactor in enzymes to perform biochemically significant functions, while controlling intracellular levels of copper with a sophisticated network of trafficking proteins. Proteins and enzymes that utilize copper as a cofactor have evolved significantly different coordination environments than copper trafficking proteins, as a result of the different functions they perform. Of particular interest was characterizing the unique Cu(I) binding events observed in some of these proteins, the extracellular N-terminal regions of eukaryotic high affinity copper transport proteins (Ctr), and the bacterial periplamsic CusF protein of the CusFBCA Cu(I)/Ag(I) efflux pathway.

Model peptides corresponding to the methionine rich binding motifs (Mets motifs) were characterized in terms of Cu(I) binding affinity, stoichiometry, and metal specificity, via an ascorbic acid oxidation assay and electrospray ionization mass spectrometry. Metal induced structural features and coordination environments were elucidated with NMR, CD and X-ray spectroscopy. A series of peptides was also examined to infer the relative Cu(I) binding affinities, and susceptibility to oxidation, of methionine, histidine, cysteine residues found in copper binding motifs. The resistance of Cu+ specific peptides to metal catalyzed oxidation is also described. Attempts were also made to model the Cu(I)/Ag(I) tryptophan cation-π interaction observed in CusF.

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