Peptide-Induced Bilayer Thinning Structure of Unilamellar Vesicles and the Related Binding Behavior as Revealed by X-ray Scattering

Abstract

To quantitatively correlate membrane thinning with peptide binding affinity, 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 bialyer thinning of the substrate-oriented multilamellar membranes (MLM) with premixed melittin. Moreover, thinning of the melittin-ULV bilayer saturates at ∼4 %, significantly lower than the critical thinning of ∼8% (determined via the correspondingly premixed peptide-MLM bilayers) for thermally equilibriated formation of membrane pores, revealing a critical influence of binding affinity for water soluble peptides. Scaling the peptide-ULV bilayer thinning 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 for 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 may have a same thinning constant upon binding of a same peptide, hence providing a basis in establishing X-ray-based binding isotherms for thermodynamic binding parameters of late stage peptide-membrane interactions prior to pore formation.