Structure determination of membrane proteins in five easy pieces

Abstract

Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment.

Figure 1. Membrane proteins that have been expressed, purified, and demonstrated to give high-resolution solid-state NMR spectra in lipid bilayers

This panel provides a series of targets for the development of NMR spectroscopy and structure determination methods. From left to right they have increasing size and complexity, ranging from a 35-residue, single transmembrane, α-helical domain, to a full-length 350-residue protein with seven transmembrane helices, and an outer membrane protein with β-barrel architecture. Solid-state NMR can be used to determine the structures of these membrane proteins in native-like membrane environments and conditions. This is demonstrated here with MerFt, a truncated construct of MerF with 60 residues and two transmembrane helices, and with CXCR1, a G-protein coupled receptor with 350 residues and seven transmembrane helices.

Figure 2. The effects of rotational diffusion on the chemical shift anisotropy powder pattern from ¹³C’ labeled membrane proteins in proteoliposomes using simulated spectra

(A, B) ¹³C’ chemical shift powder patterns for a residue in a transmembrane helix approximately parallel to the membrane normal, rotationally averaged by rotational diffusion (A) or static (B). (C, D) Same as (A, B), respectively, except that the sample is undergoing slow (5 kHz) magic angle spinning. No sideband intensity is observed in (C) because of the narrow frequency breadth of the motionally averaged powder pattern. (D) A family of sidebands is observed because of the broad frequency breadth of the static powder pattern. The sideband intensities can be used to calculate the powder pattern line shape.

Figure 3. Multi-dimensional MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in proteoliposomes

(A) Two-dimensional ¹³C/¹⁵N heteronuclear correlation spectrum of MerFt in proteoliposomes. (B) Two-dimensional ¹H-¹⁵N dipolar coupling/¹³C chemical shift SLF spectrum of MerFt in proteoliposomes. The signals for L31 and D69 can be identified in these crowded two-dimensional spectra, which have had all resonances assigned. (C, D) Two-dimensional ¹H-¹⁵N dipolar coupling/¹³C chemical shift SLF planes from a three-dimensional spectrum. The few signals in each plane demonstrate that complete resolution is achieved in the three-dimensional spectrum. The same dipolar coupling frequencies are measured in panels B, C, D for both L31 and D69. Similar results are obtained for all other residues and provide the input for the structure calculations.

Figure 4. One-dimensional slices from multi-dimensional MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled MerFt in proteoliposomes

One dimensional ¹⁵N solid-state NMR spectra of uniformly ¹⁵N/¹³C labeled MerFt in DMPC bilayers. A. Experimental ¹⁵N amide chemical shift powder pattern spectrum of L31 (essentially identical to that for D69) obtained at low temperature (10°C). B. Simulated static ¹⁵N amide chemical shift powder pattern spectrum based on the data in Figure 4A. C. Experimental rotationally averaged ¹⁵N amide chemical shift powder pattern spectrum of D69 at 25°C. D.Same as C. except for L31. E. Simulated static Pake powder pattern for ¹H-¹⁵N amide heteronuclear dipolar coupling. F. Experimental rotationally averaged ¹H-¹⁵N heteronuclear dipolar coupling for D69 at 25°C. G. Same as F. except for L3. H. Simulated static Pake powder pattern for ¹H-¹³C heteronuclear dipolar coupling. I. Experimental rotationally averaged ¹H-¹³C heteronuclear dipolar coupling powder pattern spectrum for D69 at 25°C. J. same as I. except for L31. The experimental data in the Figure were extracted from high resolution multidimensional experiments correlating ¹³C, ¹⁵N isotropic chemical shifts and either ¹H-¹⁵N/ ¹H-¹³Cα dipolar coupling or ¹⁵N chemical shift anisotropy in the third dimension (82).

Figure 5. One-dimensional slices from multi-dimensional MAS solid-state NMR spectra of uniformly ¹³C/¹⁵N labeled CXCR1 in proteoliposomes

(A) Simulated static Pake powder pattern spectrum for a ¹H-¹⁵N heteronuclear dipolar coupling of an amide group in a peptide bond. (B, C) One-dimensional rotationally averaged ¹H-¹⁵N heteronuclear powder patterns extracted from a three-dimensional ¹³C-detected ¹H-¹⁵N dipolar coupling / ¹⁵N chemical shift SLF spectrum.¹H-¹⁵N heteronuclear dipolar coupling frequencies (noted on the left side of the spectra) are associated with ¹³Cα isotropic chemical shifts (noted on the right side of the spectra).

Figure 6. Three-dimensional structure of MerFt in phospholipid bilayers

(A) Structure of MerFt determined by the RA solid-state NMR method described here. (B) Correlation plot of back-calculated ¹H-¹⁵N heteronuclear dipolar couplings from the refined structure with experimentally measured couplings. The correlation coefficient is 0.94.