Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR

Abstract

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.2 GHz proton frequency) and ultrafast MAS spinning (<100 kHz) promise to accelerate acquisition, while reducing sample requirement, both of which are critical to membrane protein studies. Here, we review recent advances in ssNMR methodology used for structure determination of membrane proteins in native and mimetic environments, as well as the study of protein functions such as protein dynamics, and interactions with ligands, lipids and cholesterol.

Fig. 1. Selected examples of membrane protein structure determination using solid-state NMR. (A) Experimental PISEMA spectrum of uniformly ¹⁵N labeled trans-membrane ion-channel domain of Vpu in bilayers aligned on glass plates and superposition of 100 calculated backbone structures of the trans-membrane helix of Vpu. (B) Cα–Cα region of a PDSD spectrum of influenza A M2 S31N with τ_mix = 400 ms recorded at ω_0H/2π = 750 MHz and ω_r/2π = 14.287 kHz. A 4-fold molar excess of rimantadine drug was present in the sample and did not perturb the chemical shifts of the S31N mutant (code 2N70). (C) Structure and proton-detected (H)NH spectra of AIkL using 60 and 100 kHz MAS. (D) Peaks from the Trp side chain region of a CP-based ¹H–¹⁵N-correlation (blue) overlaid with either the projection of the (H)CANH spectrum or an INEPT-based sequence (red) and structure of OMPA (code 5MWV). Panels A and B are reproduced with permission from the American Chemical Society. Panel C is reproduced with permission from Springer. Panel D is reproduced with permission from the Nature Publishing Group.

Fig. 2. Membrane protein structures deposited in the Protein Data Bank, which were solved by solid-state NMR. Beta barrel structures are denoted in red and helical structures are in blue.

Fig. 3. Spectra illustrating protein isotope labelling schemes. Panels A–E show different deuteration schemes for membrane protein samples, and F–H show different carbon labelling schemes. (A) Comparison of 2D ¹H–¹⁵N CP-HSQC MAS NMR spectra acquired at 305 K on (blue) fully protonated U-[¹³C,¹⁵N] proteorhodopsin in DMPC:DMPA lipids at 100 kHz MAS, and (red) U-[²H,¹⁵N,¹³C] proteorhodopsin, reprotonated in 100% protonated buffer, in lipids at 60 kHz MAS and at a field strength of 23.5 T. (B) Methyl spectra of M2 labeled with –¹³CD₂H methyl groups in the I, L, and V residues. The J-transferred 2D spectrum of the stereospecifically ¹³C²H₂¹H-methyl-labeled sample is shown in subpanel a, with assignments of isoleucine Cδ1, leucine Cδ2, and valine Cγ2 methyl groups. (C) Cut-outs of a 2D ¹³C–¹H spectrum (red) measured at 55 kHz MAS and 800 MHz (sub panel c) using a V,L,K ¹H-cloud labelling of BamA, exchanged in D₂O. A spectrum measured with fully protonated BamA (grey) is superposed. (D) ¹H-detected (H)NH spectrum of fractionally deuterated KcsA. (E) Identification of a cross beta strand contact (F99–I114 Hα) in the beta barrel membrane protein VDAC in lipid bilayers. (F) Resonance assignment of OmpG. Spectral regions of ¹³C–¹³C correlation spectra comprising Cα–Cβ cross-peaks of leucine, threonine, and histidine in three different samples using amino acid specific labelling and DARR mixing. For the peaks indicated by pink dots in these ¹³C–¹³C spectra, no strip could be found in the ¹H-detected 3D spectra. (G) ¹³C–¹³C RFDR MAS correlation spectrum of [U-¹³C,¹²C-FLY,¹⁵N] VDAC in DMPC 2D crystals. (H) Comparison of sparse ¹³C labelling patterns of rhodopsin from Leptosphaeria maculans (LR) in 1D ¹³C-CP MAS spectra. Top to bottom: U-¹³C,¹⁵N-LR, 2-¹³C-glucose-LR, and 1,3-¹³C-glucose-LR. Significant differences in labelling patterns are marked for methyl groups at 10–20 ppm, Cα of glycine at ∼47 ppm, Cα of Ala at ∼55 ppm, Cα of Leu at ∼58 ppm, Cζ of Arg or Tyr at ∼160 ppm, and carbonyl side chain atoms of Asp, Asn, Glu or Gln at ∼180 ppm. Panels A–C and G are reproduced with permission from the American Chemical Society. Panel D is reproduced with permission from Wiley. Panel E is reproduced with permission from Springer. Panel F is reproduced with permission from Nature publication group. Panel H is reproduced with permission from Springer.

Fig. 4. Signal initiated from cholesterol, lipids and water instead of protein. (A) Cholesterol labeled with ¹⁹F, ¹³C, and ²H for determining cholesterol binding to M2. ssNMR structure of Udorn M2(22–62) (PDB ID code 2L0J), (B) Water and lipid contacts shown on the homology model of AIkL using OmpW (PDB 2F1T) as a template. Lipid contacts are colored yellow and water contacts are colored blue. Residues for which no contact is observed or assigned are colored in grey. Panel A is reproduced with permission from PNAS, Panel B is reproduced with permission from Wiley.

Fig. 5. Influenza A M2 HN (left) and NC (right) spectra. Measurements were performed at 950 MHz and 55 kHz or 100 kHz MAS. The apo state is displayed in grey and the rimantadine drug bound state is displayed in red. Large chemical shift perturbations are observed upon drug binding.

Fig. 6. (A) (H)NH spectra of fully protonated Influenza A M2 at 950 MHz and 1.2 GHz spectrometers. (B) Selections from the alpha region of CP-based HC correlation spectra of CitApc. Spectra were recorded on U-[¹³C,¹⁵N] labelled CitApc reconstituted in protonated lipids. C (H)NH spectra of crystalline α-PET hVDAC. The MAS frequency was 100 kHz and about 0.5 mg of each sample was used. Measurements performed at a spectrometer frequency of 1.2 GHz (28 T B₀ field) are denoted in red and spectra measured at 950 MHz (23.5 T B₀ field) are denoted in blue. Figures are reproduced with permission from MDPI.