NMR structural studies of membrane proteins

Abstract

The three-dimensional structures of membrane proteins are essential for understanding their functions, interactions and architectures. Their requirement for lipids has hampered structure determination by conventional approaches. With optimized samples, it is possible to apply solution NMR methods to small membrane proteins in micelles; however, lipid bilayers are the definitive environment for membrane proteins and this requires solid-state NMR methods. Newly developed solid-state NMR experiments enable completely resolved spectra to be obtained from uniformly isotopically labeled membrane proteins in phospholipid lipid bilayers. The resulting operational constraints can be used for the determination of the structures of membrane proteins.

Figure 1

2D ¹H chemical shift/¹⁵N chemical shift heteronuclear correlation spectra of uniformly ¹⁵N labeled proteins in micelles using solution NMR spectroscopy (GL Veglia et al., unpublished data). (a) Magainin (25 residues) in DPC. (b) Acetylcholine M2 peptide (25 residues) in DPC. (c) Major coat protein from fd bacteriophage (50 residues) in SDS. (d) Vpu from HIV-1 (81 residues) in DHPC. (e) MerT from the bacterial mercury detoxification system (122 residues) in SDS.

Figure 2

1D ¹⁵N chemical shift solid-state NMR spectra of the ¹⁵N labeled fd coat protein in phospholipid bilayers (a–d). (e) Simulated powder pattern for a rigid ¹⁵N labeled amide site. The ¹⁵N amide chemical shift tensor elements _σ11, _σ22 and _σ33, referenced to liquid ammonia at 0 ppm, are indicated. (a) Uniformly ¹⁵N labeled coat protein in magnetically oriented bicelles tilted to the parallel orientation with the addition of lanthanide ions [42]. (b) Selectively ¹⁵N leucine-labeled coat protein in oriented phospholipid bilayers [35^••]. (c) Uniformly ¹⁵N labeled coat protein in oriented bilayers [35^••]. (d) Uniformly ¹⁵N labeled coat protein in unoriented bilayer vesicles [35^••]. The narrow resonance band centered at the isotropic frequency near 115 ppm results from backbone sites that are mobile and unstructured. The narrow peak near 30 ppm is from the amino groups of the lysine sidechains and the N terminus.

Figure 3

2D ¹⁵N chemical shift/¹H-¹⁵N heteronuclear dipolar coupling spectral planes from 2D and 3D solid-state NMR spectra of uniformly ¹⁵N labeled membrane proteins in oriented phospholipid bilayers. (a) Magainin. (b) Acetylcholine M2 peptide [5]. (c) Coat protein from the fd bacteriophage [35^••]. (d) Colicin E1 [37^••]. (a–d) are complete 2D PISEMA (polarization inversion with spin exchange at the magic angle) spectra. (e) Plane extracted from a 3D correlation spectrum of magainin at 15.7 ppm ¹H chemical shift. (f) Plane extracted from a 3D correlation spectrum of fd coat protein at 11.0 ppm ¹H chemical shift. The resonance assigned to Leu41 in the hydrophobic transmembrane helix is marked with the arrow. (g) Plane extracted from a 3D correlation spectrum of fd coat protein at 11.6 ppm ¹H chemical shift. The resonance assigned to Leu14 in the amphipathic in-plane helix is marked with the arrow.

Figure 4

Average structures of the AChR M2 peptide calculated from solution NMR distance constraints (a,b) and from the solid-state NMR orientational constraints obtained from the 2D spectrum shown in Figure 2b (c,d) [5,8^••]. Both structures are shown in the bilayer membrane in the exact orientation determined from solid-state NMR. The helix long axis is tilted approximately 12° from the membrane normal. The backbone structure of a protein can be described equivalently by vectors representing bonds between nonhydrogen atoms, as by the peptide planes formed by the individual rigid peptide bonds and their directly bonded atoms. Both representations of the M2 peptide backbone are shown. (a,c) Peptide plane representations with the N–H bonds highlighted. (b,d) Vector representation including the C–O bonds.