Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: Probing the role of hydrophobic residue size in the central region of the fusion peptide

The amino-terminal region of the membrane-anchored subunit of influenza vir us hemagglutinin, the fusion peptide, is crucial for membrane fusion of thi s virus. The peptide is extruded from the interior of the protein and inser ted into the lipid bilayer of the target membrane upon induction of a confo rmational change in the protein by low pH, Although the effects of several mutations in this region on the fusion behavior and the biophysical propert ies of the corresponding peptides have been studied, the structural require ments for an active fusion peptide have still not been defined, To probe th e sensitivity of the fusion peptide structure and function to small hydroph obic perturbations in the middle of the hydrophobic region, we have individ ually replaced the alanine residues in positions 5 and 7 with smaller (glyc ine) or bulkier (valine) hydrophobic residues and measured the extent of fu sion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers, We f ind that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectros copy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respectiv e infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant incre ases of the order parameter of the lipid hydrocarbon chains, suggesting tha t the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.