Discovery and Characterization of Two Folded Intermediates for Outer Membrane Protein TolC Biogenesis.

TolC is the outer membrane protein responsible for antibiotic efflux in E. coli. Compared to other outer membrane proteins it has an unusual fold and has been shown to fold independently of commonly used periplasmic chaperones, SurA and Skp. Here we find that the assembly of TolC involves the formation of two folded intermediates using circular dichroism, gel electrophoresis, site-specific disulfide bond formation and radioactive labeling. First the TolC monomer folds, and then TolC assembles into a trimer both in detergent-free buffer and in the presence of detergent micelles. We find that a TolC trimer also forms in the periplasm and is present in the periplasm before it inserts in the outer membrane. The monomeric and trimeric folding intermediates may be used in the future to develop a new approach to antibiotic efflux pump inhibition by targeting the assembly pathway of TolC.

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 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.

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 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.