Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans

Abstract

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.