The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other vira l fusion peptides, caused a reduction in the bilayer to hexagonal phase tra nsition temperature of dipalmitoleoylphosphatidylethanolamine (Tw), suggest ing its ability to promote negative curvature in membranes. Surprisingly, a n elongated peptide corresponding to the 33 N-terminal amino acids raised T -H, although it was more potent than the 16-amino acid fusion peptide in in ducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosp hatidylethanolamine/dioleoylphosphatidylchol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anch ored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic pote ncy of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itsel f had no fusogenic activity. The 16-amino acid N-terminal fusion peptide an d the longer 33-amino acid peptide were labeled with NBD. Fluorescence bind ing studies indicate that both peptides bind to the membrane with similar a ffinities, indicating that the increased fusogenic activity of the longer p eptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spect roscopy. We find that the amino-terminal fusion peptide is inserted into th e membrane as a P-sheet and the 17 C-terminal amino acids lie on the surfac e of the membrane, adopting an cr-helical conformation. It was further demo nstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV i nserts into the membrane and, like other viral Fusion peptides, lowers TH. Tn addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.