Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects.
The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.
Wieprecht
T
T
Department of Biophysical Chemistry, Biocenter of the University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
Apostolov
O
O
Beyermann
M
M
Seelig
J
J
D000890:Anti-Infective Agents / Q000737:chemistry*; D023181:Antimicrobial Cationic Peptides; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry*; D054806:Magainins; D009682:Magnetic Resonance Spectroscopy; D008563:Membrane Lipids / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D013816:Thermodynamics; D029867:Xenopus Proteins
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D008051:Lipid Bilayers; D054806:Magainins; D008563:Membrane Lipids; D010455:Peptides; D029867:Xenopus Proteins; C045866:peptide-Gly-Leu-amide; C053301:magainin 2 peptide, Xenopus