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Abstract

The lasso peptide is a type of ribosomally synthesized and post-translationally modified peptide (RiPP), possessing unique properties and potentially applicable to biological activity. A natural product often derived from gene clusters of actinobacteria and proteobacteria in genome mining, these peptides are typically highly stable due to their slip-knotted structure. Lasso structures have demonstrated antimicrobial properties as well as inhibitory activity, by binding receptors or enzymes. While many peptides have been studied for their metal-binding abilities, which also are relevant to their biological functions, the binding capacities of lasso peptides have yet to be explored. Astexin-3, a lasso with resistance to thermal unthreading and found in Asticcacaulis excentricus, was the chosen candidate for this work. Construction of a plasmid containing the genes encoding astexin and heterologous expression of the peptide in Escherichia coli enables further study, a process designed in previous work. The binding affinity of astexin-3 to copper(II) and iron(III) was explored because of the prevalence of these metals in living organisms. Several techniques enabled characterization of astexin-3-copper(II) complexes, including a titration curve generated using fluorescence quenching of astexin-3 with additions of the metal, utilizing the emission of the tryptophan in this peptide. The results of this work indicate that astexin-3 has potential to function as a binder of copper(II) at a pH close to that of the typical physiological pH; the peptide may therefore be useful in controlling ion concentrations within cells and have applications in pharmaceutical manufacturing of drugs. Fluorescence spectroscopy was proven to be an effective technique for measuring the dissociation constant, though other techniques in future work will be necessary to validate this finding.