PORINS OF HAEMOPHILUS-INFLUENZAE TYPE-B MUTATED IN LOOP-3 AND IN LOOP-4

Porin (341 amino acids; mass of 37,782 Da) in the outer membrane of Ha emophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da, Molecular modelin g of Hib porin identified its structural similarities to OmpF of Esche richia coli and disclosed for Bib porin a shorter length of loop 3 and a longer length of loop 4, By site-directed mutagenesis of the porin gene ompP2, mutant porins were constructed to contain 6 or 12 amino ac id deletions either in loop 3 or in surface-exposed loop 4, Wild type Hib porin and mutant porins were expressed in a nontypeable H, influen zae strain deleted for the ompP2 gene, The mutant porins were purified and reconstituted into planar bilayers, tested for channel formation and compared with wild type Bib porin, Mutant Haemophilus porin posses sing a B-amino acid deletion in loop 3 displayed a broad distribution of single channel conductance values, while deletion of 12 amino acids from the same loop destabilized the porin channel, By comparison, del etion of 6 or of 12 amino acids from loop 4 of Bib porin resulted in a n increased single channel conductance (1.15 and 1.05 nanosiemens, res pectively) compared with wild type Bib porin (0.85 nanosiemens), The C 3 epitope of the poliovirus VP1 capsid protein was inserted either int o loop 3 or into loop 4 of Bib porin, By flow cytometry, the C3 epitop e was detected as surface-exposed in strains expressing C3 insertion i n loop 4; in strains expressing C3 insertion in loop 3, the epitope wa s inaccessible, We propose that loop 4 of Bib porin, although surface- accessible, is oriented toward the central axis of the pore and that d eletions in this loop increase the single channel conductance by widen ing the pore entrance.