Solid-state nuclear magnetic resonance (NMR) spectroscopy was applied to the membrane-bound form of a synthetic peptide representing the 23-residue N-terminal fusion peptide domain of the HIV-1 gp41 envelope glycoprotein. 1D solid-state NMR line width measurements of singly 13C carbonyl labeled peptides showed that a significant population of the membrane-bound peptide is well-structured in its N-terminal and central regions while the C-terminus has more disorder. There was some dependence of line width on lipid composition, with narrower line widths and hence greater structural order observed for a lipid composition comparable to that found in the virus and its target T cells. In the more ordered N-terminal and central regions of the peptide, the 13C carbonyl chemical shifts are consistent with a nonhelical membrane-bound conformation. Additional evidence for a beta strand membrane-bound conformation was provided by analysis of 2D rotor-synchronized magic angle spinning NMR spectra of doubly 13C carbonyl labeled peptides. Lipid mixing and aqueous contents leakage assays were applied to demonstrate the fusogenicity of the peptide under conditions comparable to those used for the solid-state NMR sample preparation.

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.

The infrared spectra of the band of the 2Π–2Π asymmetric hydrogen stretching vibration in the three isotopic acetylene ions C2H2+ (ν3), 13C2H2+ (ν3), and DCCH+ (ν1) have been observed and analyzed. The high resolution infrared spectra were recorded using a difference-frequency laser spectrometer as the tunable coherent infrared source probing an ac glow discharge. Velocity modulation, noise subtraction, and unidirectional multipassing of the infrared beam through the discharge cell provided high sensitivity. C2H2+ was produced in a gas mixture of H2, He, and either CH4 or C2H2, with a total pressure of ≊7 Torr in multiple-inlet–outlet air-, water-, and liquid-nitrogen-cooled discharge tubes; C2H2 freezing precluded its use in liquid-N2-cooled discharges. Complicated by a strong perturbation whose maximum occurred at N′=15 for F1 and N′=14 for F2, the assignment of the spectrum of normal C2H2+ was made possible by (1) fortuitous discharge conditions which provided unambiguous discrimination of C2H2+ lines from among concurrent CH3+ and C2H3+ lines, and (2) fitting the ground state combination differences. Sufficiently high N transitions were observed where Λ doubling was evident. The average bond lengths rz(CH)=1.077 (5) Å and rz(CC)=1.257 (8) Å were calculated from the spectroscopic constants determined from nonlinear least-squares fitting. Vibration–rotation interactions, the Renner–Teller interaction of perturbing states, plasma chemistry, and the relevance of the work in astrophysics are discussed.

The vibration-rotation energy level pattem of protonated acetylene, C2H3+, in the v6 (C-H antisymmetric stretching) vibrational state is anomalous and irregular because of the coupling between the rotational motion and the tunneling of the three protons among their equilibrium positions. The resultant spectral anomaly and the coexistence of C-H bands of other carbocations such as CH3+, C2H2+, CH2+, etc., in our positive column discharge using He-dominated gas mixtures had made the analysis of the C2H3+ spectrum difficult. In the present paper we use a hollow cathode discharge to simplify plasma chemistry and to make a more definitive and extensive analysis of the spectrum. A 3 m hollow cathode discharge cell has been constructed with a multiple reflection optical system giving an effective path length of 30 m. A gas mixture of C2H2 and H2 with pressures of 0.03 and 1.1 Torr, respectively, has produced spectral lines of C2H3' from 3192 to 3083 cm-' which are almost completely free of those from other carbocations. The purity of the spectrum, together with the accurate ground state rotational constants recently reported by the Lille millimeter wave group, has allowed us to assign spectral lines up to J = 25 and K, = 4 and to determine extensive sets of the A-E splittings due to the proton tunneling in the excited state. An attempt has been made to analyze the plasma chemistry in the hollow cathode on the basis of earlier plasma diagnostic studies of the negative glow region. A model was used in which the primary molecular ions H2+ and C2H2+ are generated due to ionization by "hot" primary and secondary electrons. In the plasma these ions undergo ion-neutral reactions to produce H3+ and C2H3+, which are dissociated by reactions with C2H2 and recombination with "ultimate" electrons. By assuming proper number densities of primary, secondary, and ultimate electrons, semiquantitative agreement with the experimentally estimated ion densities has been obtained.

Summary The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. 2H and 31P NMR spectroscopy were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster 31P transverse relaxation and faster transverse relaxation of acyl chain 2H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader 31P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.