Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.

OmpF porin is a nonspecific pore protein from the outer membrane of Escherichia coli. Previously, a set of mutants was selected that allow the passage of long maltodextrins that do not translocate through the wildtype pore. Here, we describe the crystal structures of four point mutants and one deletion mutant from this set; their functional characterization is reported in the accompanying paper (Saint, N., Lou, K.-L., Widmer, C., Luckey, M., Schirmer, T., Rosenbusch, J. P. (1996) J. Biol. Chem. 271, 20676 -20680). All mutations have a local effect on the structure of the pore constriction and result in a larger pore cross-section. Substitution of each of the three closely packed arginine residues at the pore constriction (Arg-42, Arg-82, and Arg-132) by shorter uncharged residues causes rearrangement of the adjacent basic residues. This demonstrates mutual stabilization of these residues in the wild-type porin. Deletion of six residues from the internal loop (⌬109 -114) results in disorder of seven adjacent residues but does not alter the structure of the ␤-barrel framework. Thus, the large hollow ␤-barrel motif can be regarded as an autonomous structure.

In the homotrimeric OmpF porin from Escherichia coli, each channel is constricted by a loop protruding into the ␤-barrel of the monomer about halfway through the membrane. The water-filled channels exist in open or closed states, depending on the transmembrane potential. For the transition between these conformations, two fundamentally different mechanisms may be envisaged: a bulk movement of the constriction loop L3 or a redistribution of charges in the channel lumen. To distinguish between these hypotheses, nine mutant proteins were constructed on the basis of the highresolution x-ray structure of the wild-type protein. Functional changes were monitored by measuring single-channel conductance and critical voltage of channel closing. Structural alterations were determined by x-ray analysis to resolutions between 3.1 and 2.1 Å. Tethering the tip of L3 to the barrel wall by a disulfide bridge (E117C͞A333C), mobilizing L3 by perturbing its interaction with the barrel wall (D312N, S272A, E296L), or deleting residues at the tip of the loop (⌬116-120) did not alter appreciably the sensitivity of the channels to an external potential. A physical occlusion, due to a gross movement of L3, which would cause the channels to assume a closed conformation, can therefore be excluded.Outer membranes of Escherichia coli provide efficient protection from noxious agents in the environment, such as detergents, toxins, and proteolytic enzymes. Their asymmetrical structure, glycolipids in the outer leaflets and phospholipids in the inner, constitutes an impermeable barrier that requires diffusion pathways for the translocation of nutrients and metabolites. In Gram-negative bacteria, this task is accomplished largely by porins. In these trimeric proteins, with masses of 37,000 Da per subunit, each monomer consist of a ␤-barrel that harbors the water-filled transmembrane channel. The pathway is constricted by a loop (L3) which connects ␤-strands 5 and 6 of the barrel and protrudes into the channel lumen, narrowing it midway across the membrane to about 7 ϫ 11 Å (Fig. 1). The passage of solutes across the channel is limited to small (Ͻ600 Da), polar molecules and is slightly cation-selective in OmpF porin (4). Only a few larger solutes require specialized translocation pathways (5). We have studied the structures of this protein to high resolution in both structural and functional terms (2,3,(6)(7)(8)(9).The equilibrium between open and closed states of the channels depends on the applied voltage (7). This phenomenon is poorly understood (10), since the overall potential difference across the outer membrane has been found to be small (11). However, a significant electrostatic field exists parallel to the plane of the membrane in the constriction site (1, 6), and its constituent charges may be affected by an applied potential. This hypothesis is vindicated by the results obtained with replacements of ionizable groups by uncharged residues (2). Also, it has recently been described that polysaccharides (8), membrane-derived oligos...

TolA is an inner membrane protein with three domains: a transmembrane N‐terminus and periplasmic central and C‐terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti‐TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins.

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