A solid-state NMR index of helical membrane protein structure and topology

Abstract

The secondary structure and topology of membrane proteins can be described by inspection of two-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift polarization inversion spin exchange at the magic angle spectra obtained from uniformly (15)N-labeled samples in oriented bilayers. The characteristic wheel-like patterns of resonances observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices and hence provide direct indices of the secondary structure and topology of membrane proteins in phospholipid bilayers. We refer to these patterns as PISA (polarity index slant angle) wheels. The transmembrane helix of the M2 peptide corresponding to the pore-lining segment of the acetylcholine receptor and the membrane surface helix of the antibiotic peptide magainin are used as examples.

FIG. 1

PISEMA spectra calculated for a 19-residue α-helix with 3.6 residues per turn and uniform dihedral angles at various helix tilt angles relative to the bilayer normal. A. 0°. B. 10°. C. 20°. D. 30°. E. 40°. F. 50°. G. 60°. H. 70°. I. 80°. J. 90°. Spectra were calculated on a Silicon Graphics O2 computer (Mountain View, CA), using the FORTRAN program FINGERPRINT (21, 24). The principal values and molecular orientation of the ¹⁵N chemical shift tensor (σ₁₁ = 64 ppm; σ₂₂ = 77 ppm; σ₃₃ = 217 ppm; σ₃₃NH = 17°) and the NH bond distance (1.07 Å) were as previously determined (19).

FIG. 2

Helical wheel projection and two-dimensional PISEMA spectra of the uniformly ¹⁵N-labeled polypeptides in oriented lipid bilayers. A, B, and C. AChR M2. D, E, and F. Magainin II. A. Helical wheel projection of the amide N atoms of AChR M2. B. Experimental PISEMA spectrum of AChR M2, which provided most of the orientational constraints used for structure determination (10). C. Spectrum calculated for an α-helix with 3.6 residues per turn, identical dihedral angles, and a tilt of 12° relative to the membrane normal. D. Helical wheel view of the CA atoms of the magainin II helix with the N-terminus in front. The three-dimensional orientation of the peptide was determined from the angular constraints for Val17 derived from the three-dimensional solid-state NMR spectrum (24). The dashed line marks the boundary between polar (white) and hydrophobic (gray) residues in the amphipathic helix. E. Experimental PISEMA spectrum of magainin II. F. Spectrum calculated from the solution NMR structure of magainin in lipid micelles (PDB file 2MAG). Conditions for sample preparation and for solid-state NMR experiments of AChR M2 (10) and magainin (28) have been described. Spectra were calculated as described in Fig. 1.

FIG. 3

Correspondence between membrane protein helix tilt and polarity, and the resulting PISEMA spectra for uniformly ¹⁵N-labeled protein in oriented bilayers. A, D, G, and J. Helical wheels rotated by various values of the polar angle χ. B, E, H, and K. Helices rotated through various values of χ about their long axes (HA) and tilted by δ = 12° (B, E) and δ = 90° (H, K) away from the membrane normal (n). The y axis of the laboratory frame points out of the page. C, F, I, and L. Calculated PISEMA spectra for the various helix rotations and tilts. The NH bond vectors of the polar opposite residues 2 and 11 in the helical wheels are highlighted. The light gray areas in B, E, H, and K represent the lipid bilayer.