Temperature dependence and resonance assignment of 13C NMR spectra of selectively and uniformly labeled fusion peptides associated with membranes.
HIV-1 and influenza viral fusion peptides are biologically relevant model fusion systems and, in this study, their membrane-associated structures were probed by solid-state NMR (13)C chemical shift measurements. The influenza peptide IFP-L2CF3N contained a (13)C carbonyl label at Leu-2 and a (15)N label at Phe-3 while the HIV-1 peptide HFP-UF8L9G10 was uniformly (13)C and (15)N labeled at Phe-8, Leu-9 and Gly-10. The membrane composition of the IFP-L2CF3N sample was POPC-POPG (4:1) and the membrane composition of the HFP-UF8L9G10 sample was a mixture of lipids and cholesterol which approximately reflects the lipid headgroup and cholesterol composition of host cells of the HIV-1 virus. In one-dimensional magic angle spinning spectra, labeled backbone (13)C were selectively observed using a REDOR filter of the (13)C-(15)N dipolar coupling. Backbone chemical shifts were very similar at -50 and 20 degrees C, which suggests that low temperature does not appreciably change the peptide structure. Relative to -50 degrees C, the 20 degrees C spectra had narrower signals with lower integrated intensity, which is consistent with greater motion at the higher temperature. The Leu-2 chemical shift in the IFP-L2CF3N sample correlates with a helical structure at this residue and is consistent with detection of helical structure by other biophysical techniques. Two-dimensional (13)C-(13)C correlation spectra were obtained for the HFP-UF8L9G10 sample and were used to assign the chemical shifts of all of the (13)C labels in the peptide. Secondary shift analysis was consistent with a beta-strand structure over these three residues. The high signal-to-noise ratio of the 2D spectra suggests that membrane-associated fusion peptides with longer sequences of labeled amino acids can also be assigned with 2D and 3D methods.
Bodner
Michele L
ML
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Gabrys
Charles M
CM
Parkanzky
Paul D
PD
Yang
Jun
J
Duskin
Craig A
CA
Weliky
David P
DP
D000595:Amino Acid Sequence; D002247:Carbon Isotopes; D002784:Cholesterol / Q000737:chemistry; D015700:HIV Envelope Protein gp41 / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008563:Membrane Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013816:Thermodynamics
D002247:Carbon Isotopes; D015700:HIV Envelope Protein gp41; D008081:Liposomes; D008563:Membrane Lipids; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D002784:Cholesterol