Immobilization of the Influenza A M2 Transmembrane Peptide in Virus Envelope−Mimetic Lipid Membranes: A Solid-State NMR Investigation

Abstract

The dynamic and structural properties of membrane proteins are intimately affected by the lipid bilayer. One property of membrane proteins is uniaxial rotational diffusion, which depends on the membrane viscosity and thickness. This rotational diffusion is readily manifested in solid-state NMR spectra as characteristic lineshapes and temperature-dependent line narrowing or broadening. We show here that this whole-body uniaxial diffusion is suppressed in lipid bilayers mimicking the composition of eukaryotic cell membranes, which are rich in cholesterol and sphingomyelin. We demonstrate this membrane-induced immobilization on the transmembrane peptide of the influenza A M2 (AM2-TM) proton channel protein. At physiological temperature, AM2-TM undergoes uniaxial diffusion faster than ~ 105 s−1 in DLPC, DMPC and POPC bilayers, but the motion is slowed by two orders of magnitude, to < 103 s−1, in a cholesterol-rich virus-envelope-mimetic membrane (“viral membrane”). The immobilization is manifested as near rigid-limit 2H quadrupolar couplings and 13C-1H, 15N-1H and 13C-15N dipolar couplings for all labeled residues. The immobilization suppresses intermediate time scale broadening of the NMR spectra, thus allowing high-sensitivity and high-resolution spectra to be measured at physiological temperature. The conformation of the protein in the viral membrane is more homogeneous than in model PC membranes, as evidenced by the narrow 15N lines. The immobilization of the M2 helical bundle by the membrane composition change indicates the importance of studying membrane proteins in as native environments as possible. It also suggests that eukaryote-mimetic lipid membranes may greatly facilitate structure determination of membrane proteins by solid-state NMR.

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

“…At this stage we were interested in overall properties, and in particular the question whether nanodiscembedded CzcD undergoes fast axial rotation inside the nanodisc (which itself is immobilized because of the sedimentation brought about by MAS). Such axial rotation has been shown for several membrane proteins in liposomes [20,21]. The presence of fast axial rotation is expected to strongly reduce dipolar order parameters.…”

“…ref. [21,22]. In CzcD with its cytoplasmic domain the N-H bonds are distributed almost uniformly, such that overall rotation would result in significant reduction of the dipolar coupling.)…”

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

“…Our study employs the TM peptide reconstituted into the same eukaryotic lipid mixture that has been used for studying WT AM2 and BM2 [32, 35], which enables us to compare the structure and dynamics of the mutant with the wild-type rigorously for this conformationally plastic protein and in addition allows us to translate the structural findings to full-length M2 for which the TM peptide is a fully functional analog.…”

“…Higher membrane fluidity and negatively charged lipids favor H37 protonation [31–33]. Cholesterol promotes the α-helical conformation [34], immobilizes tetramer rotational diffusion [35], and stabilizes tetramer assembly [36, 37]. Membrane thickness affects the tilt angle of the TM helix [33, 38–40], and negative-curvature lipids can alter the tetrameric assembly of the protein [41].…”

Oligomerization state and supramolecular structure of the HIV‐1 Vpu protein transmembrane segment in phospholipid bilayers

“…1 H-13 C dipolar-shift correlation (DIPSHIFT) measurements 53,56,68 were performed in a quasithree-dimensional manner with the rf pulse sequence in Supporting Information Figure S1c, using a Varian InfinityPlus spectrometer and Varian 5.0 mm MAS NMR probe operating at 150.7 MHz 13 C NMR frequency (14.1 T magnetic field). Sample volumes were 160 lL and contained $20 mg of Vpu , with the larger volume and higher field being required for adequate signal-to-noise.…”