Self-Assembly of Catenanes from Lasso Peptides.

Lasso peptides exist naturally in a threaded state as [1]rotaxanes, and we reasoned that lasso peptides cleaved in their loop region could serve as building blocks for catenanes. Mutagenesis of the lasso peptide microcin J25 (MccJ25) with two cysteine residues followed by cleavage of the peptide with trypsin led to a [2]rotaxane structure that self-assembled into a [3]catenane and [4]catenanes at room temperature in aqueous solution. The [3]catenane represents the smallest ring size of a catenane composed solely of polypeptide segments. The NMR structure of the [3]catenane was determined, suggesting that burial of hydrophobic residues may be a driving force for assembly of the catenane structure.

Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3.

Lasso peptides are a class of knot-like polypeptides in which the C-terminal tail of the peptide threads through a ring formed by an isopeptide bond between the N-terminal amine group and a side chain carboxylic acid. The small size (∼20 amino acids) and simple topology of lasso peptides make them a good model system for studying the unthreading of entangled polypeptides, both with experiments and atomistic simulation. Here, we present an in-depth study of the thermal unthreading behavior of two lasso peptides astexin-2 and astexin-3. Quantitative kinetics and energetics of the unthreading process were determined for variants of these peptides using a series of chromatography and mass spectrometry experiments and biased molecular dynamics (MD) simulations. In addition, we show that the Tyr15Phe variant of astexin-3 unthreads via an unprecedented "tail pulling" mechanism. MD simulations on a model ring-thread system coupled with machine learning approaches also led to the discovery of physicochemical descriptors most important for peptide unthreading.