STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF OMPF PORIN MUTANTS SELECTED FOR LARGER PORE-SIZE .2. FUNCTIONAL-CHARACTERIZATION

The effects on the channel characteristics of four single amino acid s ubstitutions in OmpF porin and of a deletion mutant in the constrictio n loop L3 have been studied. These mutations are all located in the na rrow section of the channel of the protein that forms pores across the outer membrane of Escherichia coli, The single channel conductance of the deletion mutant (Delta 109-114) is decreased by one third, wherea s the point mutations do not exhibit significant deviations from that of the wild-type protein. The mutants exhibit drastic changes in ion s electivities. In the wild-type protein, the critical threshold potenti al (V-c), above which channels close reversibly, exhibits a strong pH dependence, with a titration point of similar to pH 7.7, which is abol ished in all mutants studied here. Diffusion of six monosaccharides is little affected in the point mutants, while four disaccharides are ta ken up at highly increased rates by the deletion mutant. The functiona l results, presented here, are correlated to the x-ray structures of t he mutants (Leu, K.-L., Saint, N., Prilipov, A, Rummel, G., Benson, S. A., Rosenbusch, J.P., and Schirmer, T. (1998) J. Biol. Chem. 271, 2066 9-20675). in most, but not all, cases, the structural changes explain the functional alterations observed.