Determining the depth of insertion of dynamically invisible membrane peptides by gel-phase 1H spin diffusion heteronuclear correlation NMR

Abstract

Solid-state NMR determination of the depth of insertion of membrane peptides and proteins has so far utilized 1H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid membrane. For membrane proteins or peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection. Here we show that the rigid-solid HETCOR experiment, with an additional spin diffusion period, can be used to determine the depth of proteins in gel-phase lipid membranes, where the proteins are immobilized to give high-intensity solid-state NMR spectra. Demonstration on two membrane peptides with known insertion depths shows that well-inserted peptides give rise to high lipid cross peak intensities and low water cross peaks within a modest spin diffusion mixing time, while surface-bound peptides have higher water than lipid cross peaks. Furthermore, well-inserted membrane peptides have nearly identical 1H cross sections as the lipid chains, indicating equilibration of the peptide and lipid magnetization. Using this approach, we measured the membrane topology of the α-helical fusion peptide of the paramyxovirus, PIV5, in the anionic POPC/POPG membrane, in which the fusion peptide undergoes intermediate-timescale motion at physiological temperature. The gel-phase HETCOR spectra indicate that the α-helical fusion peptide is well inserted into the POPC/POPG bilayer, spanning both leaflets. This insertion motif gives insight into the functional role of the α-helical PIV5 fusion peptide in virus-cell membrane fusion.

Conformation and Lipid Interaction of the Fusion Peptide of the Paramyxovirus PIV5 in Anionic and Negative-Curvature Membranes from Solid-State NMR

“…The resulting line broadening precludes the LC-phase 1 H spin diffusion experiment 60 for measuring the insertion depth of the peptide. Therefore, we carried out the gel-phase spin diffusion experiment, 57 which resolves the water, lipid, and peptide 1 H signals in the indirect dimension by 1 H homonuclear decoupling. Strong cross peaks between lipid protons and peptide 13 C signals indicate deep insertion of the peptide into the membrane.…”

Heating and temperature gradients of lipid bilayer samples induced by RF irradiation in MAS solid‐state NMR experiments

“…) as hydration levels increased from 30% to 50%. For the POPC liposome, because it remained in fluid phase state at all hydration levels [according to the 1 H line widths of N(CH 3 ) 3 ], hydration levels little affected the temperature elevation (Fig. ).…”

Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain

“…To investigate if the post‐AH cytoplasmic segment lies on the membrane surface, we measured the depth of insertion of M2(22–71) in a site‐specific manner using the gel‐phase 2D 1 H‐ 13 C correlation experiment [Fig. (a)] with 1 H spin diffusion mixing times of 4 and 25 ms. At 4 ms, A30 and G34 Cα peaks show much higher lipid CH 2 cross peaks than water cross peaks [Fig.…”

“…2D 15 N‐ 13 C correlation spectra were measured on a 400 MHz (9.4 Tesla) Bruker NMR spectrometer using a REDOR pulse train for polarization transfer . Gel‐phase 13 C‐ 1 H heteronuclear correlation spectra were measured under 7 kHz MAS between 248 and 258 K. The frequency‐switched Lee‐Goldburg (FSLG) sequence was used to suppress the 1 H‐ 1 H dipolar coupling during the 1 H evolution period. The 1 H spin diffusion mixing time was 4 and 25 ms, and Lee‐Goldburg cross polarization was used to transfer the 1 H magnetization to 13 C. 13 C‐ 1 H dipolar couplings were measured using the DIPSHIFT correlation experiment under 7 kHz MAS at 303 K. The FSLG sequence was used for 1 H homonuclear decoupling during t 1 .…”