Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Abstract

Clean MAS observation of 13 C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13 C-15 N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13 C carbonyl and its directly bonded 15 N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic-and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13 C, 15 N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13 C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

“…For the HFP3-8 FLG and HFP2-14 AAG samples associated with PC:PG, there were shoulders at ~178 and 179 ppm, respectively, which correlated with helical conformation of Ala, Leu, and Phe residues. These results were consistent with previous studies of the conformation of membrane-associated HFP with peptide:lipid ~ 0.04 and with previous observations of greater preference for β strand conformation in cholesterol-containing membranes (27,28,35,55,56,59,70). The data demonstrated that samples containing HFP2-14 AAG have qualitatively larger (ΔS/ S 0 ) exp than do samples containing HFP labeled at other residues.…”

Structure, Topology, and Tilt of Cell-Signaling Peptides Containing Nuclear Localization Sequences in Membrane Bilayers Determined by Solid-State NMR and Molecular Dynamics Simulation Studies

“…REDOR-filtered experiments as described in the literature (47) were utilized to suppress the 13 C background signal from lipids of MLVs. A 5mm triple-resonance Varian/Chemagnetics MAS probe was used and the sample was spun at a speed of 8 kHz spinning speed at −20°C.…”

Structure and Orientation of Pardaxin Determined by NMR Experiments in Model Membranes

“…For the S 1 acquisition, a 15 N 180°pulse at the middle and end of each rotor period in the dephasing time was applied. Other details of the REDOR filtering experiment can be found elsewhere (28). Spectra of DMPC MLVs containing 3 mol % pardaxin suggest that the 13 C-labeled site ( 13 CЈ-Leu) has an isotropic chemical shift value (referenced relative to tetramethylsilane) in the range characteristic of residues in an ␣-helical conformation (10,29,30).…”