A STUDY OF BOBENZOXY-D-PHENYLALANINE-L-PHENYLALANINE-GLYCINE, AN INHIBITOR OF MEMBRANE-FUSION, IN PHOSPHOLIPID-BILAYERS WITH MULTINUCLEAR MAGNETIC-RESONANCE

The anti-viral and membrane fusion inhibitor, carbobenzoxy-D-phenylala nine-L-phenylala(ZfFG), was studied in phospholipid bilayers, where ea rlier studies had indicated this peptide functioned. Multinuclear magn etic resonance (NMR) studies were performed with isotopically labeled peptide. A peptide labeled in the glycine carboxyl with C-13 was synth esized, and the isotropic C-NMR chemical shift of that carbon was meas ured as a function of pH. A pK(a) of 3.6 for the carboxyl was determin ed from the peptide bound to a phosphatidylcholine bilayer. ZfFG inhib its the formation by sonication of highly curved, small unilamellar ve sicles. Experiments as a function of pH revealed that this ability of ZfFG was governed by a pK(a) of 3.7. Therefore the protonation state o f the carboxyl of ZfFG appeared to regulate the effectiveness of this anti-viral peptide at destabilizing highly curved phospholipid assembl ies. Such destabilization had previously been discovered to be related to the mechanism of the anti-fusion and anti-viral activity of this p eptide. The location of the carboxyl of ZfFG in the membrane was probe d with paramagnetic relaxation enhancement of the C-13 Spin lattice re laxation of the carboxyl carbon in the glycine of ZfFG (enriched in C- 13). Results suggested that this carboxyl is at or above the surface o f the phospholipid bilayer. The dynamics of the molecule in the membra ne were examined with H-2-NMR studies of ZfFG, deuterated in the alpha -carbon protons of the glycine. When ZfFG was bound to membranes of ph osphatidylcholine, a sharp H-2-NMR spectral component was observed, co nsistent with a disordering of the glycine methylene segment of the pe ptide. When ZfFG was bound to N-methyl dioleoylphosphatidylethanolamin e (N-methyl DOPE) bilayers at temperatures below 30 degrees C, a large quadrupole splitting was observed. These results suggest that ZfFG li kely inhibits membrane fusion from the surface of the lipid bilayer, b ut not by forming a tight, stoichiometric complex with the phospholipi ds.