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 Haemophilus influenzae type b (Hib) permits diffusion into the periplasm of small solutes up to a molecular mass of 1400 Da. Molecular modeling of Hib porin identified its structural similarities to OmpF of Escherichia coli and disclosed for Hib 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 acid deletions either in loop 3 or in surface-exposed loop 4. Wild type Hib porin and mutant porins were expressed in a nontypeable H. influenzae 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 Hib porin. Mutant Haemophilus porin possessing a 6-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, deletion of 6 or of 12 amino acids from loop 4 of Hib porin resulted in an increased single channel conductance (1.15 and 1.05 nanosiemens, respectively) compared with wild type Hib porin (0. 85 nanosiemens). The C3 epitope of the poliovirus VP1 capsid protein was inserted either into loop 3 or into loop 4 of Hib porin. By flow cytometry, the C3 epitope was detected as surface-exposed in strains expressing C3 insertion in loop 4; in strains expressing C3 insertion in loop 3, the epitope was inaccessible. We propose that loop 4 of Hib porin, although surface-accessible, is oriented toward the central axis of the pore and that deletions in this loop increase the single channel conductance by widening the pore entrance.
Srikumar
R
R
Department of Microbiology and Immunology, McGill University Montreal, Québec H3A 2B4, Canada.
Dahan
D
D
Arhin
F F
FF
Tawa
P
P
Diederichs
K
K
Coulton
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli; D005434:Flow Cytometry; D006193:Haemophilus influenzae / Q000737:chemistry / Q000235:genetics*; D007163:Immunosorbent Techniques; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D008051:Lipid Bilayers; D018272:Porins