Investigations into the polymorphism of lipid A from lipopolysaccharides of Escherichia coli and Salmonella minnesota by Fourier-transform infrared spectroscopy

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Abstract
The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5° and 58°C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41°, 46°, 44° and 47°C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4′-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52°C for E. coli lipid A). The analysis of >CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22°, 30°, 27° and 25.5°C (first transition) and at about 34°, 42°, 38.5° and 40.5°C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: 1 Up to about 30°C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of >CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions. 2 At elevated temperatures no straightforward interpretation of the data was possible: while the X-ray data did not indicate any drastic changes in the bilayer arrangement at the molecular level even after completion of the acyl chain melting process, the fluorometric and calorimetric data suggested the existence of more complex supermolecular arrangements at temperatures above the main transition.

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