In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K‐12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding

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Abstract
The trimeric protein LamB of Escherichia coli K‐12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane. Each monomer consists of an 18‐stranded antiparallel β‐barrel with nine surface loops (L1 to L9). The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro. In vivo, single‐, ΔL4, ΔL5, ΔL6, and double‐loop deletions, ΔL4 + ΔL5 and ΔL5 + ΔL6, abolished maltoporin functions, but not the double deletion ΔL4 + ΔL6 and the triple deletion ΔL4 + ΔL5 + ΔL6. While deletion of the central variable portion of loop L9 (ΔL9v) affected maltoporin function only moderately, the combination of ΔL9v with the double deletion of loops L4 and L6 (triple deletion ΔL4 + ΔL6 + ΔL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro. In vitro, the carbohydrate‐binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes. The deletion of loop L9v alone (LamBΔL9v), of two loops L4 and L6 (LamBΔL4 +ΔL6), of three loops L4, L5 and L6 (LamBΔL4 +ΔL5 + ΔL6) or of L4, L6 and L9v (LamBΔL4 + ΔL6 +ΔL9v) had relatively little influence on the carbohydrate‐binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side. The deletion of one of the loops L4 (LamBΔL4) or L6 (LamBΔL6) resulted in an asymmetric carbohydrate binding. The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non‐reducing end in advance. The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on‐rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side.

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