Biophysical characterisation of lysozyme binding to LPS Re and lipid A

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide ( LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The beta<->alpha gel to liqu id crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with poly myxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with s ynchrotron radiation X-ray diffraction and the binding stoichiometry with m icrocalorimetry. The results were compared with those found with zwitterion ic and negatively charged phospholipids. It can clearly be shown that lysoz yme binds electrostatically to charged groups of the endotoxin molecules wi th the consequence of acyl-chain rigidification and an initiation of a tran sition from inverted cubic to multilamellar structures. The binding stochio metry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and li pid A, indicating a dominant binding of lysozyme to-the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by th e use of a dephospho LPS, Parallel to Lysozyme binding to endotoxin, a conf ormational change of the secondary structure in the protein from mainly alp ha helix to more unordered structures takes place, while the residual beta- sheet substructure does not exhibit a clear concentration dependence. Bindi ng is found to be specific for the endotoxins since, for the zwitterionic p hosphatidylcholine, no binding is observed and, fur the negatively charged phosphatidylglycerol, only very weak binding is found. The results are disc ussed in the context of the ability of lysozyme to reduce endotoxicity.