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 liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stoichiometry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and lipid 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 the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly alpha helix to more unordered structures takes place, while the residual beta-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.
Brandenburg
K
K
Forschungszentrum Borstel, Center for Medicine and Biosciences, Germany. kbranden@fz-borstel.de
Koch
M H
MH
Seydel
U
U
D002151:Calorimetry; D004731:Endotoxins / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D008050:Lipid A / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry*; D009113:Muramidase / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012475:Salmonella / Q000737:chemistry / Q000235:genetics*; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D013696:Temperature; D014961:X-Ray Diffraction
D004731:Endotoxins; D005782:Gels; D008050:Lipid A; D008070:Lipopolysaccharides; D010710:Phosphates; D010743:Phospholipids; D011135:Polysaccharides, Bacterial; D009113:Muramidase