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ABSTRACT Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called thebarrelassemblymachine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of theEscherichia coliOmpC protein into the OMin vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called thebarrelassemblymachine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues inEscherichia coliOmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane. 

Recent studies in polymer physics have created macro-scale analogs to solute microscopic polymer chains like DNA by inducing diffusive motion on a chain of beads. These bead chains have persistence lengths of O(10) links and undergo diffusive motion under random fluctuations like vibration. We present a bead chain model within a new stochastic forcing system: an air fluidizing bed of granular media. A chain of spherical 6 mm resin beads crimped onto silk thread are buffeted randomly by the multiphase flow of grains and low density rising air “bubbles”. We “thermalize” bead chains of various lengths at different fluidizing airflow rates, while X-ray imaging captures a projection of the chains’ dynamics within the media. With modern 3D printing techniques, we can better represent complex polymers by geometrically varying bead connections and their relative strength, e.g., mimicking the variable stiffness between adjacent nucleotide pairs of DNA. We also develop Discrete Element Method (DEM) simulations to study the 3D motion of the bead chain, where the bead chain is represented by simulated spherical particles connected by linear and angular spring-like bonds. In experiment, we find that the velocity distributions of the beads follow exponential distributions rather than the Gaussian distributions expected from polymers in solution. Through use of the DEM simulation, we find that this difference can likely be attributed to the distributions of the forces imparted onto the chain from the fluidized bed environment. We anticipate expanding this study in the future to explore a wide range of chain composition and confinement geometry, which will provide insights into the physics of large biopolymers.