Evolutionary engineering a larger porin using a loop-to-hairpin mechanism

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane {beta}-barrels, duplication is visible with {beta}-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane {beta}-barrels. Specifically, the topology of some 16- and 18-stranded {beta}-barrels appear to have evolved through a loop to {beta}-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded {beta}-barrel and an evolutionarily related 16-stranded {beta}-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane {beta}-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to {beta}-hairpin transition. HighlightsO_LIWe find evidence supporting a novel diversification mechanism in membrane {beta}-barrels C_LIO_LIThe mechanism is the conversion of an extracellular loop to transmembrane {beta}-hairpin C_LIO_LIA chimeric protein modeling this mechanism folds stably in the membrane C_LIO_LIThe chimera has more {beta}-structure and a larger pore, consistent with a loop-to-hairpin transition C_LI