Screening, large-scale production, and structure-based classification for cystine-dense peptides

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. Their exploitation was hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression screening and large-scale production of these peptides. 46 were successfully produced in multimilligram quantities, and over 600 more were deemed expressible by stringent screening criteria. Many showed extreme resistance to temperature, proteolysis, and/or reduction, and all displayed inhibitory activity against at least one of 20 ion channels tested, confirming biological functionality. Crystal structures of 12 were determined, confirming proper cystine topology, and the utility of crystallography for studying these molecules, but highlighted the need for rational classification. Previous attempts at categorization have focused on limited subsets siloed around distinct motifs. Stepping back, we present a global definition, classification, and analysis of over 700 structures of cystine-dense peptides, unifying these molecules.