Solid state nuclear magnetic resonance studies of the influenza fusion peptide associated with membrane bilayers.

摘要

Membrane fusion in influenza virus is caused by the influenza viral hemagglutinin protein (HA). The amino terminus of the HA2 domain of the hemagglutinin protein is known as the 'fusion peptide.' In this work, solid-state nuclear magnetic resonance (NMR) spectroscopy was used to understand the structures of synthetic peptides based on the influenza fusion peptide. Because the free fusion peptide has been shown to induce fusion of liposomes in a pH-dependent way similar to the complete protein, the information gained by the study of the free peptide should be valuable for understanding the mechanism of influenza viral fusion by the influenza fusion protein.;The fusion peptide was synthesized with a solubilizing 'host-sequence'. The physical properties and biological activities of these peptides were studied to provide evidence that the peptide structures examined were relevant to the larger fusion protein system.;Because the influenza fusion peptide has been studied with a variety of lipid compositions using a number of sample preparation methods, the effects of lipid composition, peptide:lipid ratio, temperature, and pH on the membrane-bound fusion peptide structure were studied. REDOR subtraction was used to filter out the large natural abundance 13C carbonyl signals from the NMR spectra, and chemical shift was used as a convenient indicator of local secondary structure.;The data revealed at least two distinct structures of the membrane bound fusion peptide. The structure is dependent on the lipid and cholesterol composition of the membrane to which the peptide is associated. It was also shown that the fusion peptide causes pH-dependent lipid mixing regardless of its equilibrium structure. This shows that there are at least two distinct structures of the influenza fusion peptide that can induce fusion.;A proposed 310 helix proposed to be present in the low pH peptide in DPC detergent solution and in POPC:ROPG membranes but absent at neutral pH was probed by REDOR spectroscopy. Our REDOR measurements show that this structural feature is present in fusion peptide bound to DPC micelles and to POPC:POPG membranes at both low and neutral pH. Chemical shift measurements support this conclusion.