Properties of the major outer membrane protein from Neisseria gonorrhoeae incorporated into model lipid membranes.

Abstract

The major outer membrane protein from Neisseria gonorrhoeae was incorporated into artificial planar bilayer membranes by a detergent-dilution procedure. The integrated protein forms voltage-dependent aqueous pores with a minimal pore diameter estimated to be 11 A. A pore of this size suggests a role for this protein in macromolecular sieving at the level of the outer membrane. This protein self-associates preferentially in triplets of three equal unit conductance steps of 130 pS (in 0.1 M NaCl) each. The two-state model may be applied to explain the voltage-dependent conductance. The average lifetime of the open state of single channels is strongly dependent on the applied voltage, the channels shifting to the closed state at higher voltages. The pore is anion selective, differing from porins of other Gramnegative bacteria studied so far but resembling the voltage-dependent anion-selective channel of the outer membrane of mitochondria.The outer membranes of a number of Gram-negative bacteria contain highly organized passive diffusion pathways for macromolecules between 600 and 3,000 daltons (Da) (1). Porin, the protein responsible for these molecular sieving properties, has been characterized for several different types of bacteria (1-14). Porin from Escherichia coli is available now in crystalline forms amenable to ultrastructural studies (15). Porins from E. coli and Salmonella typhimurium have been incorporated into model lipid membranes (1,3,(11)(12)(13)(14). Membranes containing porin develop aqueous channels with pore sizes large enough to account for the observed permeability of the bacteria to certain nonelectrolytes (1). Crosslinking (4, 5), ultrastructural (6, 7), physicochemical (8,9), and electrophysiological (10-14) studies have indicated that porin molecules preferentially aggregate as stable trimers.Similar to that of other Gram-negative bacteria, the outer membrane of Neisseria gonorrhoeae contains a major protein (or protein I) that by itself accounts for more than 60% by weight of the protein in the outer membrane (16,17). The molecular mass of this protein is strain dependent and lies between 32 and 39 kDa (17). Previous studies have shown that the outer membranes of N. gonorrhoeae are more permeable to macromolecules than are those of E. coli and other wild-type Gram-negative bacteria (18,19). The relationship of protein I to the permeability of the gonococcal outer membrane is not clear.In this report, we have incorporated the purified gonococcal protein I into high-resistance planar lipid bilayer membranes by a simple procedure that may be applicable to other integral membrane proteins. The high resolution of electrical measurements achieved on planar bilayers allows the dissection of molecular properties associated with permeability changes conferred by channel-forming proteins. We present evidence here that the major outer membrane protein from N. gonorrhoeae is a pore-forming protein that may be responsible for the permeability properties of the outer membrane of this bacterium....

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies

“…Such homology suggests a conserved three-dimensional structure of the proteins in accord with a highly conserved functional role as a porin (Young et al, 1983;Lynch et al, 1983) and also perhaps in interactions with host cells (Blake, 1985). The similarity between the deduced amino acid sequences suggests that antigenic differences between strains must largely result from sequence variations in localized regions.…”

Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers

“…In between these two states the amount of porin channels being open or closed depends on the voltage applied. Lakey and Pattus (1989) as well as Morgan et al (1990) have also detected asymmetry in channel gating while with PhoE of E. coli and protein I of N. gonorrhoeae perfect symmetry of channel closing was observed (Dargent et al, 1986;Young et al, 1983).…”

“…Interestingly the voltages required for channel closing were different even for identical porins in the various studies. This applies to OmpF (Lakey and Pattus, 1989;Morgan et al, 1990) and OmpC Biihler, 1991) of E. coli as well as to protein I of Neisseria gonorrhoeae (Young et al, 1983 ;Mauro et al, 1988). A revealing comparison of the effects resulting from the different techniques of membrane formation was given by Lakey and Pattus (1989), but the prerequisites for channel closing at low potentials have not been investigated systematically so far.…”

Genetics of protein I of Neisseria gonorrhoeae: construction of hybrid porins.

“…Apart from its well-documented role as an anion-selective porin (1)(2)(3), PI is expressed on the surface of all gonococci, is highly antigenic in vivo (4), and has the interesting ability to insert into cell and artificial membranes directly from gonococcal cells (5,6). Compared to certain other highly variable surface-exposed components of the gonococcus-notably, pilin (7), protein II (8), and LOS (lipooligosaccharide) (9)-PI is antigenically stable in vitro.…”