Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595

Abstract

The structural polymorphism of deep rough mutant lipopolysaccharide—in many biological systems the most active endotoxin—from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg 2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the β ↔ α gel to liquid crystalline acyl chain melting. The onset, midtemperature T c, and completion of the β ↔ α phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The β ↔ α acyl chain melting temperature T c lying at 30°C at high water content (95%) increases with decreasing water content reaching a value of 50°C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg 2+. In the lower water concentration range (<50%) at temperatures below 70°C, only lamellar structures can be observed independent of the Mg 2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70°C strongly depends on the [LPS]:[Mg 2+] ratio. For large [LPS]:[Mg 2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg 2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg 2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80°C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.