Specific Binding of Ro 09-0198 (Cinnamycin) to Phosphatidylethanolamine: A Thermodynamic Analysis

Abstract

Ro 09-0198 (cinnamycin) is a tetracyclic peptide antibiotic that is used to monitor the transbilayer movement of phosphatidylethanolamine (PE) in biological membranes during cell division and apoptosis. The molecule is one of the very rare examples where a small peptide binds specifically to a particular lipid. In model membranes and biological membranes containing phosphatidylethanolamine, Ro 09-0198 forms a 1:1 complex with this lipid. We have measured the thermodynamic parameters of complex formation with high sensitivity isothermal titration calorimetry and have investigated the structural consequences with deuterium and phosphorus solid-state NMR. Complex formation is characterized by a large binding constant, K0, of 107 to 108 M-1, depending on the experimental conditions. The reaction enthalpy, ΔH°, varies between zero at 10 °C to strongly exothermic −10 kcal/mol at 50 °C. For large vesicles with a diameter of ∼100 nm, ΔH° decreases linearly with temperature and the molar heat capacity of complex formation can be evaluated as = −245 cal/mol, indicating a hydrophobic binding mechanism. The free energy of binding is ΔG° = −10.5 kcal/mol and shows only little temperature dependence. The constancy of ΔG° together with the distinct temperature-dependence of ΔH° provide evidence for an entropy−enthalpy compensation mechanism: at 10 °C, complex formation is completely entropy-driven, at 50 °C it is enthalpy-driven. Varying the PE fatty acid chain-length between 6 and 18 carbon atoms produces similar binding constants and ΔH° values. Addition of Ro 09-0198 to PE containing bilayers eliminates the typical bilayer structure and produces 2H- and 31P-NMR spectra characteristic of slow isotropic tumbling. This reorganization of the lipid matrix is not limited to PE but also includes other lipids.