Evolutionary Engineering a Larger Porin Using a Loop-to-Hairpin Mechanism

Abstract

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 β-barrels, duplication is visible with β-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 β-barrels. Specifically, the topology of some 16- and 18-stranded β-barrels appear to have evolved through a loop to β-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded β-barrel and an evolutionarily related 16-stranded β-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane β-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 β-hairpin transition.