Lasso Peptides: Exploring the Folding Landscape of Nature’s Smallest Interlocked Motifs

14 September 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Lasso peptides are a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from non-threaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically-favored unfolded and non-threaded structures, suggesting de novo lasso folding is rare. However, by modifying the ring forming residues with the appendage of thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.


lasso peptides
peptide folding
peptide synthesis
molecular dynamics
de novo folding
protein folding

Supplementary materials

Supporting Information
Simulation methods; algorithm descriptions and equations; parametrization protocol; cluster analyses; secondary structure analyses; Glu8 Ψ dihedral angle distributions; number of prelasso and pre-tadpole structures across different force fields; contact map analyses; 2-D probability distributions for multiple replicas; distribution of filtered structures based on the distance 1N-8Cδ vs. LAMBDA; total number of structures for each simulation and the ratio between pre-lassos and pre-tadpoles; cluster and secondary structure analyses for MOD1; 2-D energy maps for the MOD1 and MOD2 with His5+; contact analyses for His5; Tables S1-S2; and Figures S1-S12 (PDF).


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