Gram-negative outer-membrane proteins with multiple β-barrel domains

Outer-membrane beta barrels (OMBBs) are found in the outer membrane of gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel β-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8 to 36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers, and some function only in this state. Using a combination of sensitive sequence comparison methods and coevolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include eight-stranded barrels prevail. These multibarrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multibarrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis, suggests that their functions are not essential but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for functions beyond those of the individual barrels.

Gram-negative outer membrane proteins with multiple β-barrel domains

Outer membrane beta barrels (OMBBs) are found in the outer membrane of Gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel β-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8-36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers and some function only in this state. Using a combination of sensitive sequence-comparison methods and co-evolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include 8-stranded barrels prevail. These multi-barrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multi-barrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis suggests that their functions are not essential, but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for new functions beyond those of the individual barrels.

Redesigning OmpA Loops Using Canonical Outer Membrane Protein Loop Structures

ABSTRACTProtein loops can be difficult to design and predict. There have been multiple different algorithms developed to predict the structure of loops. Outer membrane proteins are all beta barrels and these barrels have a variety of well-documented loop conformations. Here we test three different algorithms to predict the structure of outer membrane protein loops. We find the PETALS algorithm is superior for this purpose. We then experimentally test the effect of replacing the long loops of outer membrane protein OmpA with twelve shorter designed loops. Though we succeeded in creating the smallest known outer membrane barrel, we find that the designed loops do not have a strong effect on OmpA folding.

Building bigger beta-barrels

The range of barrel-shaped proteins found in the outer membrane of certain bacteria evolved through multiple pathways.

Evolution of Environmentally-Enforced, Repeat Protein Topology in the Outer Membrane

Outer membrane beta barrels (OMBBs) are the proteins on the surface of Gram negative bacteria. These proteins have diverse functions but only a single topology, the beta barrel. It has been suggested that this common fold is a repeat protein with the repeating unit of a beta hairpin. By grouping structurally solved OMBBs by sequence, a detailed evolutionary story unfolds. A strand-number based pathway manifests with progression from a primordial 8-stranded barrel to 16-stranded and then to 18-stranded barrels. The transitions from 16- to 18-stranded barrels show mechanisms of strand number variation without domain duplication, such as a loop to hairpin transition. This indicates that repeat protein topology can be perpetuated without genetic duplication likely because the topology is being enforced by the membrane environment. Moreover, we find the evolutionary trace is particularly prominent in the C-terminal half of OMBBs which may be relevant to understanding OMBB folding pathways.

Modeling Beta-Traces for Beta-Barrels from Cryo-EM Density Maps

Cryo-electron microscopy (cryo-EM) has produced density maps of various resolutions. Althoughα-helices can be detected from density maps at 5–8 Å resolutions,β-strands are challenging to detect at such density maps due to close-spacing ofβ-strands. The variety of shapes ofβ-sheets adds the complexity ofβ-strands detection from density maps. We propose a new approach to model traces ofβ-strands forβ-barrel density regions that are extracted from cryo-EM density maps. In the test containing eightβ-barrels extracted from experimental cryo-EM density maps at 5.5 Å–8.25 Å resolution,StrandRollerdetected about 74.26% of the amino acids in theβ-strands with an overall 2.05 Å 2-way distance between the detectedβ-traces and the observed ones, if the best of the fifteen detection cases is considered.

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

In vitro folding studies of outer membrane beta-barrels have been invaluable in revealing the lipid effects on folding rates and efficiencies as well as folding free energies. Here, the biophysical results are summarized, and these kinetic and thermodynamic findings are considered in terms of the requirements for folding in the context of the cellular environment. Because the periplasm lacks an external energy source the only driving forces for sorting and folding available within this compartment are binding or folding free energies and their associated rates. These values define functions for periplasmic chaperones and suggest a biophysical mechanism for the BAM complex.