N-15 NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insert ion into target membranes of a stretch of amino acids located at the N-term inus of the HA, subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using N-15 NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-gl ycero-3-phosphocholine). The high value of this pK(a) is indicative of stab ilization of the protonated form of the amine group through noncovalent int eractions. The shift reagent Pr3+ had large effects on the N-15 resonance f rom the cr-amino group of Gly(1) of the fusion peptide in DOPC vesicles, in dicating that the terminal amino group was exposed to the bulk solvent, eve n at low pH. Furthermore, electron paramagnetic resonance studies on the fu sion peptide region of spin-labeled derivatives of a larger HA construct ar e consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N -terminal of the fusion peptide is close to the aqueous phase and is proton ated. Thus neither a change in the state of ionization nor a significant in crease in membrane insertion of this group is associated with increased fus ogenicity at low pH.