The Formation of Ordered structures of Bacterial Porins in a Lipid Bilayer and the Analysis of their Morphology by Atomic Force Microscopy

详细

Ordered nanostructures of porin from the outer membrane of Yersinia pseudotuberculosis (YpOmpF) were formed in two ways: from proteoliposomes and by direct protein reconstitution in the pre-deposited phospholipid bilayer on mica surface. The morphological analysis of the structures was performed by atomic force microscopy. It was shown that the efficiency of formation, the degree of homogeneity, and the size of porin domains substantially depend on the experimental conditions and the presence of lipopolysaccharide in a porin sample or in the bilayer. It was found that using proteoliposomes resulted in formation of the aggregates of porin nanodomains on the mica surface, with uneven distribution in the bilayer and quite different size ranges (50–250 nm). In the case of direct reconstruction of porin, it was shown that a decrease in pH of the solubilizing buffer promotes the inclusion of a sufficiently large amount of protein as homogeneous domains with an average size of 35–40 nm but does not lead to the formation of extended nanostructured regions in the bilayer. The most efficient incorporation of porin into the lipid bilayer with the formation of clusters of tightly packed protein domains was achieved using a porin sample in combination with peptidoglycan and lipopolysaccharide, which this protein is tightly bound to in the native bacterial membrane.