Unified free energy landscapes of μ-conotoxins reveal prefolding predictors of folding pathway classification

Conotoxins are short, disulfide-rich peptide toxins produced by cone snails. Their strong affinity for receptors involved in neuromuscular transmission makes them promising therapeutic leads. While at least one conotoxin-based analgesic has been brought to market to date, a greater understanding of the overall subclasses of short disulfide-rich peptides is needed so that the entire class may be developed as a search space rather than a pool of single therapeutic candidates.

The μ conotoxin class exists in a folding spectrum from ordered to disordered--hirudin-like to BPTI-like. In hirudin-like folding, formation of disulfides occurs indiscriminately followed by reshuffling, leading to an ensemble of kinetically trapped isomers. In BPTI-like folding, formation of disulfides occurs through step-by-step formation of the native disulfide bonds. Here, we employ the composite diffusion map approach to study the unified free energy surface of pre-folded equilibrium. We show that in the absence of the disulfides the conotoxins can be thought of as disordered polymers. We identify the most important collective modes of the unified folding landscape and demonstrate that diffusion distance from the projected native state is a predictor of where the conotoxins fall on the folding continuum.  Overall, this work sheds important light on the folding processes and free energy landscapes of different μ-conotoxins and represents an important step in design space exploration.