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.

Outer membrane protein evolution.

Outer membrane proteins have remarkably homogeneous structure. They are all up down ß-barrels. Up down barrels themselves are composed of repeated sets of ß-hairpins. The consistency of the usage of the ß-hairpin throughout the outer membrane milieu allows for interrogation of the evolution of these repetitive structures. Here we describe recent investigations of outer membrane protein evolution and how evolutionary precepts have been used for novel outer membrane protein design.

Evolutionary pathways of repeat protein topology in bacterial outer membrane proteins.

Outer membrane proteins (OMPs) are the proteins in the surface of Gram-negative bacteria. These proteins have diverse functions but a single topology: the ß-barrel. Sequence analysis has suggested that this common fold is a ß-hairpin repeat protein, and that amplification of the ß-hairpin has resulted in 8-26-stranded barrels. Using an integrated approach that combines sequence and structural analyses, we find events in which non-amplification diversification also increases barrel strand number. Our network-based analysis reveals strand-number-based evolutionary pathways, including one that progresses from a primordial 8-stranded barrel to 16-strands and further, to 18-strands. Among these pathways are mechanisms of strand number accretion without domain duplication, like a loop-to-hairpin transition. These mechanisms illustrate perpetuation of repeat protein topology without genetic duplication, likely induced by the hydrophobic membrane. Finally, we find that the evolutionary trace is particularly prominent in the C-terminal half of OMPs, implicating this region in the nucleation of OMP folding.

Outer membrane protein design.

Membrane proteins are the gateway to the cell. These proteins are also a control center of the cell, as information from the outside is passed through membrane proteins as signals to the cellular machinery. The design of membrane proteins seeks to harness the power of these gateways and signal carriers. This review will focus on the design of the membrane proteins that are in the outer membrane, a membrane which only exists for gram negative bacteria, mitochondria, and chloroplasts. Unlike other membrane proteins, outer membrane proteins are uniquely shaped as ß-barrels. Herein, I describe most known examples of membrane ß-barrel design to date, focusing particularly on categorizing designs as: Firstly, structural deconstruction; secondly, structural changes; thirdly, chemical function design; and finally, the creation of new folds.