Applications of NMR to membrane proteins

Abstract

Membrane proteins present a challenge for structural biology. In this article, we review some of the recent developments that advance the application of NMR to membrane proteins, with emphasis on structural studies in detergent-free, lipid bilayer samples that resemble the native environment. NMR spectroscopy is not only ideally suited for structure determination of membrane proteins in hydrated lipid bilayer membranes, but also highly complementary to the other principal techniques based on X-ray and electron diffraction. Recent advances in NMR instrumentation, spectroscopic methods, computational methods, and sample preparations are driving exciting new efforts in membrane protein structural biology.

Keywords: Bilayer; Lipid; Membrane protein; NMR; Solid-state NMR.

Figure 1. Examples of membrane protein structures determined in detergent micelles by solution NMR

The PDB file numbers are listed below each structure. (A) Mitochondrial voltage dependent anion channel VDAC [31]. (B) Archaeal photo sensory rhodopsin pSRII with bound retinal (orange) [33]. (C) Bacterial inner membrane protein DsbB with bound quinone (orange) [34]. (D) Channel domain of influenza BM2, showing His, Trp and Ser side chains (sticks) lining the pore [40]. (E) Transmembrane domain ζ-ζ dimer of the T cell receptor CD3, showing side chains (sticks) that mediate dimerization [37]. (F) Mitochondrial stannin showing the metal-binding Cys-Trp-Cys motif (sticks) at the membrane surface [36] (G) Human Na,K-ATPase regulator FXYD1 showing side chains (sticks) and Gly CA atoms (spheres) that mediate intra-membrane association with the Na,K-ATPase α subunit [38].

Figure 2. Structure of Y. pestis outer membrane protein Ail and effect of detergent on its ligand binding activity

(A) Solution NMR structure of Ail, determined in DePC micelles, showing Arg and Lys side chains (yellow) at the membrane surface [74]. (B) Solution NMR ¹H/¹⁵N TROSY spectra of Ail in 170 mM DePC (black) or nanodiscs (red). (C) Fibronectin binding activity of Ail analyzed by enzyme linked immunosorbent assay, where increasing concentrations of Ail are added to fibronectin-coated plates. Adapted from [73].

Figure 3. Examples of membrane protein structures determined in detergent-free phospholipids by solid-state NMR

The PDB file numbers are listed below each structure. (A) Anabaena sensory rhodopsin with bound retinal (yellow) [111]. (B) human chemokine receptor, CXCR1 [112]. (C) M2 ¹H channel from influenza virus showing His and Trp side chains (sticks) lining the channel pore [113]. (D) Bacterial inner membrane protein DsbB [115,116]. (E) Mycobacterium cell division protein, CrgA showing side chains (sticks) that mediate intra-membrane helix-helix association [117]. (F) Membrane-inserted form of the fd bacteriophage coat protein showing polar side chains (sticks) in the N-terminal helix exposed to bulk water [118].

Figure 4. Solid-state NMR spectra of uniformly ¹⁵N labeled FXYD2 in magnetically oriented phospholipid bilayers

(A–D) 2D ¹H/¹⁵N PISEMA) and 1D ¹⁵N OS solid-state NMR spectra of FXYD2 in lipid bilayers aligned with the membrane perpendicular (A, B) or parallel (C, D) to the magnetic field. Peaks from the transmembrane helix (TM) trace wheel-like patterns (red circles). Peaks assigned to Arg guanidinium NH groups are prominent (blue boxes). (E) Solution NMR structure of FXYD2 determined in micelles, showing the bundle of ten lowest energy structures. The positions of amide N atoms (blue spheres) were restricted by experimental plane distance restraints as described [132]. Adapted from [132].

Figure 5. 2D ¹³C-¹³C spectrum of (¹⁵N, ¹³C, ²H) Ail in liposomes

(A) Fingerprint region of the DARR (Dipolar Assisted Rotational Resonance) spectrum showing examples of resolved signals from Ala, Ile, Ser, Thr, Val residues (yellow boxes). (B) Expanded spectral region showing examples of short-range intra-residue correlations (blue), short-range inter-residue correlations (green), and long-range inter-residue correlations (red). (C) Structure of Ail in micelles (PDB: 2N2L) showing inter-strand connections (red) assigned in the DARR spectrum. The spectrum was obtained at 900 MHz, at 10°C, with 200 ms DARR mixing time, and 144 scans per increment. Adapted from [144].

Figure 6. 2D ¹H-¹⁵N correlation spectra of Ail in phospholipid bilayers

(A) Solid-state NMR ¹H-detected CP-HSQC spectrum of (¹⁵N, ¹³C, ²H) labeled Ail in liposomes, recorded at 900 MHz, 30°C, with 160 scans and a MAS rate of 60 kHz. (B) Solution NMR ¹H-detected TROSY spectrum of (¹⁵N,¹³C,²H) labeled Ail in nanodiscs prepared with ²H labeled lipids, recorded at 800 MHz, 45°C, with 128 scans. Adapted from [144].

Figure 7. The EEFx potential improves structure calculations of Anabaena sensory rhodopsin in lipid bilayers

Structures of monomeric ASR were calculated using solid-state NMR distance and dihedral angle restraints. Bar plots represent results for: the crystal structure (PDB 1xio; pink) [181]; the average for ten models in the ensembles of the deposited solid-state NMR structure (PDB 2m3g; red) [111]; the ensemble calculated with the standard simple repulsive potential of Xplor-NIH (gray); or the ensemble calculated with the EEFx potential of Xplor-NIH (blue) [173]. (A) Agreement with the PDB crystal structure (PDB 1xio) evaluated as average pairwise RMSD of atomic coordinates. (B) Precision evaluated as average pairwise RMSD of atomic coordinates in each ensemble. (C) MolProbity score evaluation of the four structures; note this is a cost: the lower the better. (D) Comparisons of the crystal structure (PDB 1zxio, pink) with the lowest energy structure generated with EEFx (blue). The horizontal lines depict the boundaries of the 25 Å thick EEFx membrane. Adapted from [173].