Peptide-induced bilayer thinning structure of unilamellar vesicles and the related binding behavior as revealed by X-ray scattering

We have studied the bilayer thinning structure of unilamellar vesicles (ULV) of a phospholipid 1,2-dierucoyl-sn-glycero-3-phosphocholine (di22:1PC) upon binding of melittin, a water-soluble amphipathic peptide. Successive thinning of the ULV bilayers with increasing peptide concentration was monitored via small-angle X-ray scattering (SAXS). Results suggest that the two leaflets of the ULV of closed bilayers are perturbed and thinned asymmetrically upon free peptide binding, in contrast to the centro-symmetric bilayer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer associates closely with peptide concentration in solution and saturates at ~ 4%, compared to the ~ 8% maximum thinning observed for the correspondingly premixed peptide-MLM bilayers. Linearly scaling the thinning of peptide-ULV bilayers to that of the corresponding peptide-MLM of a calibrated peptide-to-lipid ratio, we have deduced the number of bound peptides on the ULV bilayers as a function of free peptide concentration in solution. The hence derived X-ray-based binding isotherm allows extraction of a low binding constant of melittin to the ULV bilayers, on the basis of surface partition equilibrium and the Gouy–Chapman theory. Moreover, we show that the ULV and MLM bilayers of di22:1PC share a same thinning constant upon binding of a hydrophobic peptide alamethicin; this result supports the linear scaling approach used in the melittin-ULV bilayer thinning for thermodynamic binding parameters of water-soluble peptides.