title: 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 (1)H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid bilayer. 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peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection.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 (1)H 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 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.
authors.affiliation: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: T
authors.initials: T
authors.lastname: Wang
authors.forename: H
authors.initials: H
authors.lastname: Yao
authors.forename: M
authors.initials: M
authors.lastname: Hong
mesh_terms: D003903:Deuterium; D008565:Membrane Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000737:chemistry*
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D003903:Deuterium