Probing the Structure of Novel Lasso Peptides

Abstract

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides that are mostly found in Proteobacteria and Actinobacteria. Lasso peptides are naturally found in gene clusters, containing a precursor peptide as well as associated maturation and transport proteins. In addition, these clusters can be inserted into plasmids for heterologous expression in E. coli, enabling additional studies on the effects of modifications of the amino acid sequences. This thesis had two aims centered around the unique structure of lasso peptides. The first was the structural analysis of novel lasso peptides by nuclear magnetic resonance spectroscopy. This was achieved through the solved structure of the lasso peptide, citrocin. The determination of the structure of a second lasso peptide, pandonodinΔC14, was also attempted, but a threaded structure could not be resolved, where the most probable cause was low water solubility. The second goal was to use a lasso peptide as a building block for a new catenane through the expression of variants of the native lasso peptide sequence. One of the variants, ubonodin G28C, containing a substitution for cysteine at the C-terminal residue was expressed at high enough levels for large-scale production and analysis. This variant may afford the possibility of C-terminal modification in the future. Several other variants of the lasso peptide, ubonodin, were expressed towards the formation of a catenane, but none had expression levels high enough for large-scale production. However, a few of these ubonodin variants still provided useful information. The relative expression levels of two variants, ubonodin Y26F and ubonodin Y27F, was informative towards the importance of steric lock residues. The higher expression level of ubonodin Y26F suggested that the C-terminal residue of the steric lock, as compared to the N-terminal residue, was more important in maintaining the threaded structure of native ubonodin. The results of this study contribute to the library of solved lasso peptide structures and to the scope of amino acid substitutions tolerated by the maturation enzymes of ubonodin.