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 ß-barrels, duplication is visible with ß-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 ß-barrels. Specifically, the topology of some 16- and 18-stranded ß-barrels appear to have evolved through a loop to ß-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded ß-barrel and an evolutionarily related 16-stranded ß-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane ß-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 ß-hairpin transition.
Subject(s)
Bacterial Outer Membrane Proteins , Porins , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Directed Molecular Evolution , Porins/chemistry , Porins/genetics , Protein Domains , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Protein Folding , Protein Conformation, beta-StrandABSTRACT
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 ß-barrels, duplication is visible with ß-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 ß-barrels. Specifically, the topology of some 16- and 18-stranded ß-barrels appear to have evolved through a loop to ß-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded ß-barrel and an evolutionarily related 16-stranded ß-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane ß-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 ß-hairpin transition. Highlights: We find evidence supporting a novel diversification mechanism in membrane ß-barrelsThe mechanism is the conversion of an extracellular loop to transmembrane ß-hairpinA chimeric protein modeling this mechanism folds stably in the membraneThe chimera has more ß-structure and a larger pore, consistent with a loop-to-hairpin transition.
