Isothermal Titration Calorimetry Studies of the Binding of a Rationally Designed Analogue of the Antimicrobial Peptide Gramicidin S to Phospholipid Bilayer Membranes

Abstract

The binding of the positively charged antimicrobial peptide cyclo [VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3- [phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptideinduced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a onesite binding model. The measured endothermic binding enthalpy (ΔH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (ΔG) of binding remains relatively constant (−8.5 to −11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TΔS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.The binding of water-soluble peptides or proteins to lipid membranes is an important issue in many biological processes. There are, for instance, several types of amphipathic peptides, including gramicidin S (GS), 1 that bind to the bacterial membrane and act as antibiotics (1, † Supported by operating grants from the Canadian Institutes of Health Research (R.N.M.), major equipment grants from the Alberta

Infectious disease: Connecting innate immunity to biocidal polymers

“…The temperature induced variation in absorbance at 440 nm for DPPC vesicles interacting with low concentration of melittin (1 mol %) indicated changes both pretransition and broadening of the main phase transition of the lipid bilayer. Dynamic light scattering (DLS) can be used to measure fusion of LUVs in the presence of the antimicrobial cyclic peptide [215]. The average hydrodynamic diameter of pure and peptide bound LUVs can be evaluated and use to determine whether vesicle fusion occurs.…”

Identification, characterization and activation mechanism of a tyrosine kinase of Bacillus anthracis

“…The conformational enthalpy is generally considered to be exothermic, as formation of secondary structure is favourable. Similarly, interaction enthalpy is also exothermic, as it involves the formation of new noncovalent interactions such as electrostatic attraction, van der Waals interactions, and hydrogen bonds [29]. However, as the overall enthalpy change is endothermic, these two terms may be negligible, and the endothermic enthalpy of association may be contributed by large positive solvation enthalpy that arises due to the release of ordered water molecules from the McsB-McsA interface (i.e.…”

“…ITC experiments were performed using a MicroCal VP-ITC-type microcalorimeter (MicroCal Inc.) at 25°C [26][27][28][29]. Temperature equilibration prior to experiments was allowed for 1-2 h. All solutions were thoroughly degassed before use by stirring under vacuum.…”

Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody–ligand interaction thermodynamics and kinetics

“…These have included protein-protein [8, 9], protein-lipid [10–12], protein-small molecule [13, 14], and protein/small molecule-DNA binding [15–17], as well as protein-carbohydrate binding [18, 19]. ITC has been used to determine the equilibrium constant, reaction stoichiometry, and changes in enthalpy, entropy and Gibbs free energy involved in biological binding events [12].…”