Characterization of Porins from the Outer Membrane of Salmonella typhimurium 2. Physical Properties of the Functional Oligomeric Aggregates

Abstract

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41 000 and 37 500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer . dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer . dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in fl-structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability-conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming pcrmeability channels in the outer membrane of Salmonella typhimurium.The outer membrane of gram-negative bacteria allows a transmembrane diffusion of small hydrophilic substances of molecular weights less than 600 [1,2]. This selective permeability of the outer membrane was restored in the artificial vesicle membranes reconstituted from phospholipids, lipopolysaccharide and the outer membrane proteins [3] and the active components for channel formation were identified as proteins with molecular weights around 35 000, called 'porin' [4,5]. Since the permeation of the solute mediated by the porin showed no requirement for metabolic energy, little temperature dependency and poor stereospecificity for the solute, we have proposed that the permeation via this protein was mediated through waterfilled 'channels' [4].In our previous work, we isolated large aggregates of porin, called complexes I and 11, as active fractions for channel formation [4]. But the monomeric form of the porin, when produced by heating complex I or I1 in the presence of dodecylsulfate, no longer retained permeability-conferring activity in reconstituted ves-~~ ~~~~

Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

“…6). These findings contrast reports on a number of porins characterized to date (31,32,37,38) including PorB class 2 protein (7) that describe superimposable CD spectra for porins incubated with either nonionic/zwitterionic detergents or SDS at concentrations as high as 1% and temperatures below 60°C. Moreover, the tertiary structure elements of class 3 are severely affected in the presence of this detergent, as monitored by tyrosyl exposure employing second derivative UV analysis (Fig.…”

“…The far UV CD spectrum ( Fig. 2A) reveals that class 3 protein retains the basic secondary structural features characteristic of other porins from E. coli (31), Salmonella typhimurium (32), and meningococcal PorB class 2 protein (7). The minima observed at 217 nm may be correlated with the native conformation of class 3 protein which is comprised predominantly of ␤-pleated sheet structures.…”

Burkholderia (formerly Pseudomonas) cepacia porin is an oligomer composed of two component proteins

“…Purification of the OmpF oligomer from E. coli B was carried out by the method described by Tokunaga et al (1979).…”

Influence of Immunopotentiators on the Antiporin Immunoglobulin G Subclass: Distribution and Protective Immunity Against Murine Salmonellosis

“…It was found to be thermostable [6], protease resistant [7] and an essential component for survival of the bacteria [8]. Hence, porin might be an ideal cell-surface component for the development of a subunit vaccine for Salmonellosis.…”