Specificity of diffusion channels produced by lambda phage receptor protein of Escherichia coli.

Abstract

The lamB protein, the receptor for phage X, was purified from the outer membrane of Escherichia coli K-12 by extraction with Triton X-100 and EDTA, chromatography on DEAE-Sephacel in Triton X-100, exchange of Triton for cholate by gel filtration, and chromatography on Sephacryl S-200 in cholate, NaCI, and EDTA. The purified protein appeared to exist as several oligomeric species. In an equilibrium retention assay with reconstituted vesicles containing phospholipids and lipopolysaccharide, the lamB protein conferred permeability for disaccharides. In a liposome swelling assay designed to measure rates of diffusion, the lamB protein conferred permeability to phospholipid liposomes for a variety of substrates. The rates obtained indicate the permeation facilitated by the lamB protein is specific, discriminating among substrates by both size and configuration. For example, maltose diffused into liposomes 40 times faster than sucrose, about 8 times faster than cellobiose, and about 12 times faster than maltoheptaose. The results suggest that the lamB protein forms a transmembrane channel containing a site (or sites) that loosely interacts with the solutes.The outer membrane of Gram-negative bacteria has several pathways for the translocation of small molecules (reviewed in ref. 1). The passage of most small, hydrophilic molecules is accomplished by porins, which form nonspecific water-filled pores. Other outer membrane proteins are involved in the transport of specific nutrients, but the mechanism of the specific transport is not understood.The A phage receptor protein, product of the lamB gene (2), is required in Escherichia coli for the transport of maltose at micromolar concentrations and of maltodextrins of higher molecular weight (3). Because direct binding studies have failed to detect an affinity of lamB protein for maltose (4), the hypothesis that it also forms a fairly nonspecific pore has been attractive. Conductivity studies on black lipid membranes containing the lamB protein give evidence for a pore of fairly large diameter (5). Furthermore, while the current work was in progress, a paper by Nakae (6) appeared in which he claimed that membranes reconstituted from phospholipids, lipopolysaccharides, and lamB protein are nonspecifically permeable to all sugars tested except stachyose. In order to reconcile this apparent lack of specificity in reconstituted systems with the specificity for maltose and maltodextrins observed in intact cells, it has been proposed that the lamB protein produces a totally nonspecific channel, and that the specificity is derived entirely from the interaction of periplasmic maltose-binding protein with the lamB protein (7).To examine the function of the lamB protein in vitro, we developed a purification scheme that utilized neither sodium dodecyl sulfate (NaDodSO4) nor the organic solvents used in earlier works (5-8). These agents could alter the activities of the protein by partially denaturing it, so we wanted to employ milder treatments to extract and purify the prot...

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…several OprD residues belonging to loops 3 and 7 and a number of backbone residues create an asymmetric charge distribution and participate in the formation of the pore-constriction zone. some mutated residues have previously been reported in carbapenem-resistant isolates and are located in or near loops 6 and 7 (rEFs 60,61 [63][64][65] . A small aliquot of liposomes is rapidly mixed in a polymer-free solution that contains the molecule of interest and that has the same osmotic pressure.…”

“…surprisingly few physico-chemical measurements have been performed to characterize the permeation of substrates across membrane channels. so far, porin permeation has been characterized qualitatively using liposome-swelling assays [63][64][65] (FIG. 2).…”

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

“…Protein a is a minor outer membrane protein with approximately the same electrophoretic mobility as PhoE protein [10]. LamB protein functions as a pore with substrate specificity for maltose and maltodextrins [32]. Molecular weights of the relevant proteins are between 47000 (for LamB protein) [33] and 35000 (for OmpA protein) [34].…”

Identification of a new porin, RafY, encoded by raffinose plasmid pRSD2 of Escherichia coli

“…In contrast to the general porins, it contains specific sugarbinding sites that facilitate diffusion of oligosaccharides through the pore (4,14,15). Moreover, the maltoporin also enhances the uptake of mono-and disaccharides (e.g., glucose and trehalose) at low substrate concentrations.…”