Solid-state NMR and membrane proteins

Abstract

The native environment for a membrane protein is a phospholipid bilayer. Because the protein is immobilized on NMR timescales by the interactions within a bilayer membrane, solid-state NMR methods are essential to obtain high-resolution spectra. Approaches have been developed for both unoriented and oriented samples, however, they all rest on the foundation of the most fundamental aspects of solid-state NMR, and the chemical shift and homo- and hetero-nuclear dipole-dipole interactions. Solid-state NMR has advanced sufficiently to enable the structures of membrane proteins to be determined under near-native conditions in phospholipid bilayers.

Keywords: Bilayers; Chemical shift anisotropy; Dipolar coupling; Magic angle spinning; Phospholipids; Structure determination.

Figure 1

The effects of rotational motion on dipole-dipole coupling and chemical shift anisotropy powder patterns. A. Theoretical line shapes for a nuclear pair with spin ½ when stationary and when in motion about an axis perpendicular to the internuclear axis. From reference [9]. B. ¹⁹F powder spectra of silver trifluoroacetate at 107°K (.) and 40°K (-). The spectra are characteristic of rotating (107°K) and rigid (40°K) CF₃ groups. From reference [10].

Figure 2

High-resolution solid-state 13C NMR spectra of a single crystal of durene. From reference [15].

Figure 3

Dipolar-decoupled natural abundance ¹³C NMR spectra of some solids obtained using single Hartmann-Hahn cross-polarization contacts of 1 ms duration. The cross-polarization spectra, obtained both with and without magic-angle spinning, are compared to some standard Fourier transform ¹³C NMR spectra of various materials in solution. Each spectrum is 8 kHz wide (at 22.6 MHz). The magnetic field increases from left to right. From reference [24].

Figure 4

Solid-state NMR spectrum of an oriented sample of polyethylene with its long axis parallel to the direction of the magnetic field. From reference [31].

Figure 5

(top) Schematic drawings of three membrane proteins aligned in planar “long-chain lipid”: Triton X-100 (q=5) bilayers. A.–C. One-dimensional solid-state ¹⁵N NMR spectra of uniformly ¹⁵N-labeled proteins. D.–F. One-dimensional solid-state ³¹P NMR spectra of the phospholipids in the same samples. A, D. The membrane-bound form of the 46-residue Pf1 coat protein. B, E. The 78-residue mercury transport protein MerE. C, F. The 350-residue G-protein-coupled receptor CXCR1. From reference [38].

Figure 6

Pure chemical shift spectrum (top) and SLF spectrum (bottom) for ¹³C in a single crystal of calcium formate. From reference [18].

Figure 7

Two-dimensional SLF spectrum of uniformly ¹⁵N-labeled Pf1 coat protein in DMPC:Triton X-100 bilayers obtained at 700 MHz. From reference [38].

Figure 8

Schematic three-dimensional and cross-sectional views of the “Fluid mosaic model” of globular membrane proteins that are completely or partially embedded within a lipid matrix. Modified from reference [52].

Figure 9

¹³C solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes. The majority of resonance intensity centered near 175 ppm is from ¹³C′ backbone sites. A. Spectrum simulated for a single ¹³C′ group in a transmembrane helix undergoing rotational diffusion around the lipid bilayer normal. B. As in Panel A except for a static ¹³C′ group in a peptide bond. The family of sidebands in panel B (red) would be observed under slow (5 kHz) MAS. C. and D. Experimental spectra obtained for a stationary sample when the protein undergoes fast rotational diffusion about the phospholipid bilayer normal C. or where the protein is immobile on the time scale of the static ¹³C′ CSA powder pattern D. E. and F. Experimental spectra obtained from a sample undergoing slow (5 kHz) MAS where the ¹³C′ CSA powder pattern is motionally averaged E, or at where a family of sidebands spanning the width of the static ¹³C′ CSA powder pattern F is observed in the absence of protein rotational diffusion. Comparisons of the powder pattern frequency breadth (A vs. B; C vs. D) or the presence of spinning sidebands (E vs. F) are diagnostic for the presence of fast rotational diffusion of the protein under the experimental conditions used to measure the CSA and DC powder patterns. From reference [58].

Figure 10

Examples of spectroscopic data for residue L31 obtained from MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in DMPC proteoliposomes at 25°C: A. two-dimensional ¹H-¹⁵N dipolar coupling/¹³C shift SLF spectrum, B. two-dimensional ¹H-¹⁵N dipolar coupling/¹³C shift SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹⁵N chemical shift frequency of 118.6 ppm. C. Two-dimensional ¹H-¹³CA dipolar coupling/¹³C shift SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹⁵N chemical shift frequency of 118.6 ppm. D. Two-dimensional ¹H-¹³CA dipolar coupling/¹H-¹⁵N dipolar coupling SLF spectral plane selected from a three-dimensional spectrum at an isotropic ¹³C chemical shift frequency of 54.6 ppm. All three spectral planes are associated with residue L31. The dashed line traces the correlations among the frequencies, which were obtained from three separate experiments. The dipolar coupling frequencies in the spectra correspond to the perpendicular edge frequencies of the corresponding powder patterns. Panel B shows that the ¹H-¹⁵N dipolar coupling motionally averaged powder pattern for L31 has a perpendicular edge frequency of 4.7 kHz, corresponding to a splitting of 9.4 kHz, and a dipolar coupling value of 18.8 kHz. From reference [58].

Figure 11

Orientation of the peptide plane associated with residue L31 based on the experimental data shown in Figure 10. From reference [85].

Figure 12

The three-dimensional structure of MerF is shown as a ribbon diagram in aqua. Both termini of the protein are in the cytosol. The structure was calculated using the data from reference [64].