Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D 2O

Abstract

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D 2O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D 2O-ESEEM intensities decrease from TOAC 1 to TOAC 8 and TOAC 16 but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC 16 is located in the bilayer leaflet opposite to that of TOAC 1 and TOAC 8. Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D 2O-ESEEM intensity is greatest for TOAC 8, indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.