The cell wall porin of Nocardia farcinica: biochemical identification of the channel‐forming protein and biophysical characterization of the channel properties

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Abstract
A channel‐forming protein was identified in cell wall extracts of the Gram‐positive, strictly aerobic bacterium Nocardia farcinica. The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine‐containing SDS–PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion‐permeable channels. The channels had on average a single‐channel conductance of 3.0 nS in 1 M KCl, 10 mM Tris‐HCl, pH 8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single‐channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single‐channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4–1.6 nm. Channel‐forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water‐filled channels that contain negative point charges.