Electron spin resonance probing of lipopolysaccharide domains in the outer membrane of Escherichia coli

Abstract

(1) Scatchard analysis of the binding of the cationic electron spin resonance probe 4-(dodecyldimethylammonium)-1-oxyl-2,2,6,6-tetramethylpiperdine bromide to lipopolysaccharide and to phospholipid indicated that this probe has a 5 fold greater affinity for anionic lipopolysaccharide than for phospholipid. In the intact outer membrane this cationic probe likely associates with the lipopolysaccharide-containing outer monolayer. (2) The temperature dependence of the mobility of the cationic spin probe in the outer membrane indicates that a structural transition occurs at 9°C in the outer monolayer. (3) A similar 9°C transition was detected in the outer membrane using the spin probe 5-doxyl stearate. This anionic probe has been shown to preferentially partition into the phospholipid enriched domains of the outer membrane. (4) A porin-lipopolysaccharide-peptidoglycan complex probed with the cationic probe also was shown to undergo a thermally induced structural change at 9°C. (5) Purified lipopolysaccharide, however, was shown to have structural transitions at 20°C and 40°C. (6) It is proposed that a structural rearrangement of the intact outer membrane occurs at approx. 9°C in both the lipopolysaccharide and phospholipid domains of the outer membrane. Furthermore, this structural transition appears to be highly dependent on lipid-protein interactions. A second thermotropic transition that occurs in the outer membrane at approx. 40 to 42°C appears to result mainly from changes in the lipopolysaccharide domain structure.