%0 Journal Article
%T Combined thermal and carboxypeptidase Y stability assays for probing the threaded fold of lasso peptides.
%A Hegemann JD
%J Methods Enzymol
%V 663
%N 0
%D 2022
%M 35168788
%F 1.682
%R 10.1016/bs.mie.2021.09.011
%X Lasso peptides are natural products belonging to the superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). The defining characteristic of lasso peptides is their threaded structure, which is reminiscent of a lariat knot. When working with lasso peptides, it is therefore of major importance to understand and evidence their threaded folds. While the full elucidation of their three-dimensional structures via NMR spectroscopy or crystallization remains the gold standard, these methods are time-consuming, require large quantities of highly pure lasso peptides, and therefore might not always be applicable. Instead, the unique properties of lasso peptides in context of their behavior at elevated temperatures and toward carboxypeptidase Y treatment can be leveraged as a tool to investigate and evidence the threaded lasso fold using only minute amounts of compound that does not need to be purified first. This chapter will provide insights into the thermal stability properties of lasso peptides and their behavior when treated with carboxypeptidase Y in comparison to a branched-cyclic peptide with the same amino acid sequence. Furthermore, it will be described in detail how to set up a combined thermal and carboxypeptidase Y stability assay and how to analyze its outcomes.