New insights into the phase behaviour of a complex anionic amphiphile: architecture and dynamics of bacterial deep rough lipopolysaccharide membranes as seen by FTIR, X-ray, and molecular modelling techniques

Abstract

Using the “functional group probing capability” of FTIR together with X-ray and molecular modelling techniques, the dynamic properties, conformational structure and phase behaviour of deep rough bacterial lipopolysaccharide (Re-LPS) have been investigated. Re-LPS (Na-salt form, pH 7) exhibited a pronounced L β→L α-like main phase transition at a mid-point temperature T m of 29°C. Only lamellar phases were observed between pH 4 and 10, in the temperature range from 5 to 70°C and upon addition of divalent cations like Ca 2+. On the basis of these findings, it is concluded that the described inverted hexagonal, or inverted micellar-like phases play no significant role under physiological conditions. The observation of precipitating aggregates at low pH and on addition of divalent cations and the pronounced dependence of phase transition temperatures and enthalpies on pH is interpreted to be due to the process of charge neutralization leading to mixed phases of strongly interacting multilamellar aggregates and rigidified polar head group regions and to the reduction of cooperativity of the acyl chain melting process. Depending on time of isothermal annealing at low temperatures, several sub-transition-like features were observed which revealed the coexistence of slightly differing acyl chain packing prior to the gel to liquid crystalline (L β→L α) transition, including hexagonal and distorted hexagonal types of packing patterns. During the main phase transition, evidence for the breakdown of distinct associates or aggregates of Re-LPS molecules was obtained. Even at elevated temperatures, fairly far above T m, complex rearrangements within the polar head group and the interfacial region accompanied by an extra increase in acyl chain disorder were observed, indicating specific LPS-LPS interactions. The comparison of theoretical and molecular modelling results with the experimental data leads to a new understanding of the peculiarities of this complex amphiphile which enable it to stabilize the asymmetric configuration of the bacterial outer membrane and its pronounced permeation barrier towards hydrophobic agents. There are several determinants for the specific functional role of LPS: (i) the high negative charge density and the conformational rigidity (especially in the polar backbone and interfacial region); (ii) the effectively reduced length of acyl-chain segments which participate in the cooperative chain melting process; (iii) the high tendency to form strongly interacting aggregates within monolamellar or multilamellar supramolecular architectures which is favoured by divalent cation bridging; and (iv) the remarkably extended interfacial region of high electron density between the hydrophilic and hydrophobic parts of the molecule.