Abstract
Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) where the C-terminal tail is threaded and sterically trapped within an N-terminal macrolactam ring. In the present work, the thermal unthreading process of the lasso peptide syanodin I was characterized using liquid chromatography, trapped ion mobility spectrometry, electron-capture dissociation, and tandem mass spectrometry (LC-TIMS-q-ECD-ToF MS/MS) and steered molecular dynamics (SMD). The analytical workflow allows for the separation (LC and TIMS) and identification (ECD MS/MS) of syanodin I kinetic intermediates as a function of the solution temperature. The syanodin WT with the plug residue Gln13 can be identified from the branched-cyclic topology based on unique retention time (RT), IMS bands, and hydrogen migration events near the molecular knot (ci•/zj' fragments). After heat treatment for 1 h at 95 °C, the unthreaded syanodin I (branched-cyclic) and an intermediate lasso structure, with Leu15 as plug residue, are identified based on the RT, IMS bands and signature ECD MS/MS fragments. Mutagenesis experiments, substituting to bulkier ([L15W] and [L15W/A16W]) and smaller ([L15A]) residues, confirmed the intermediate plug residue at Leu15. Changes in the initial plug residue Gln13 to a smaller residue ([Q13A]) resulted in a lasso structure with Leu15 as the plug residue. SMD simulations supported a loop-pulling mechanism, but also the possibility of a tail-pulling mechanism, in good agreement with the experimental observations. This is the first report of a two-step, lasso thermal unfolding mechanism driven by the two steric constraints based on loop-pulling of the lasso tail.