C 13 – C 13 and N15–C13 correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: Amino acid-type assignments and evidence for multiple conformations

Abstract

Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an approximately 20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform (13)C, (15)N labeling over large contiguous regions of amino acids. Two-dimensional (13)C-(13)C and (15)N-(13)C spectra were obtained for the membrane-bound fusion peptides and an amino acid-type (13)C assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the (13)C chemical shifts were more consistent with beta strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with beta strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous (13)C and (15)N assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Calpha nuclei. These sets of two shifts may indicate two beta strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4 T instrument, one (13)C-(13)C correlation spectrum was obtained on a 16.4 T instrument and was better resolved than the comparable 9.4 T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.