Magainin 2 amide interaction with lipid membranes: calorimetric detection of peptide binding and pore formation.

Abstract

The interaction of the antibiotic magainin 2 amide (M2a) with lipid bilayers was investigated with high-sensitivity titration calorimetry. The enthalpy of transfer of the cationic M2a to negatively charged small unilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (75:25, mol/mol) was measured as delta H = -17.0 +/- 1 kcal/mol of peptide. The adsorption isotherm was determined by injecting lipid vesicles into peptide solutions at low peptide concentrations (cPo < 7 microM). The apparent partition coefficient was Kapp approximately 1.2 x 10(4) M-1 at a peptide equilibrium concentration of 1 microM but decreased with increasing peptide concentration. The hydrophobic partitioning of M2a into the lipid membrane is modulated by electrostatic effects that arise from the attraction of the positively charged peptide to the negatively charged membrane. Using the Gouy-Chapman theory to correct for electrostatic attraction, the experimental binding isotherms can be explained with an intrinsic (hydrophobic) partition coefficient of K = 55 +/- 5 M-1 and an effective peptide charge of z = 3.7-3.8. The free energy of binding is delta G = -4.8 kcal/mol. At peptide concentrations cPo > approximately 7 microM, a second effect comes into play, and the titration enthalpies can no longer be explained exclusively by peptide partitioning. The first few injections produce enthalpies of reaction which are distinctly smaller than expected from a pure partition equilibrium, followed by a series of injections with reaction heats larger than expected. After subtracting the enthalpic contribution due to partitioning, the residual enthalpies are endothermic for the first few injections, and exothermic for the consecutive steps. Furthermore, the endothermic excess heat is compensated exactly by the exothermic excess heat; i.e., the excess heat consumed in the first part of the titration experiment is returned during the second part. Endothermic excess enthalpies are observed for total molar peptide-to-lipid ratios of P/L > approximately 3.0%, whereas exothermic excess heats were seen for 0.7% < P/L < 3.0%. Below P/L < approximately 0.7%, the binding follows the partition equilibrium. Based on earlier spectroscopic evidence, it is suggested that magainin 2 amide binds to the lipid membrane and forms pores at high peptide-to-lipid ratio, this process being characterized by an endothermic reaction enthalpy. Pore formation is reversed with increasing lipid concentration, and the peptide pores disintegrate. The limiting peptide-to-lipid ratio deduced from titration calorimetry for M2a pore formation is in excellent agreement with spectroscopic methods. The enthalpy of pore formation amounts to delta H = +6.2 +/- 1.6 kcal/mol peptide or delta H approximately 25-45 kcal/mol pore if the pore is comprised of 4-7 peptide molecules.