Efficient Segmental Isotope Labeling of Integral Membrane Proteins for High-Resolution NMR Studies

Abstract

High-resolution structural NMR analyses of membrane proteins are challenging due to their large size, resulting in broad resonances and strong signal overlap. Among the isotope labeling methods that can remedy this situation, segmental isotope labeling is a suitable strategy to simplify NMR spectra and retain high-resolution structural information. However, protein ligation within integral membrane proteins is complicated since the hydrophobic protein fragments are insoluble, and the removal of ligation side-products is elaborate. Here, we show that a stabilized split-intein system can be used for rapid and high-yield protein trans-splicing of integral membrane proteins under denaturing conditions. This setup enables segmental isotope labeling experiments within folded protein domains for NMR studies. We show that high-quality NMR spectra of markedly reduced complexity can be obtained in detergent micelles and lipid nanodiscs. Of note, the nanodisc insertion step specifically selects for the ligated and correctly folded membrane protein and simultaneously removes ligation byproducts. Using this tailored workflow, we show that high-resolution NMR structure determination is strongly facilitated with just two segmentally isotope-labeled membrane protein samples. The presented method will be broadly applicable to structural and dynamical investigations of (membrane-) proteins and their complexes by solution and solid-state NMR but also other structural methods where segmental labeling is beneficial.

Figure 1

Intein-mediated protein trans-splicing for segmental isotope labeling of membrane proteins for NMR studies. (a) Strategy to obtain properly folded membrane proteins in detergent micelles and nanodiscs by protein trans-splicing. (b) Prototype membrane proteins OmpX (2m06.pdb) and MPV17 (Alphafold model) with a β-barrel and α-helical topology, respectively. The N- and C-exteins and the splicing sites are indicated. MSP, membrane scaffold protein; MP, membrane protein.

Figure 2

High-yield production of membrane proteins by intein-mediated protein trans-splicing. (a) SDS-PAGE of the splicing reaction of OmpX. (b) ESI-MS data of the splicing reaction indicating the generation of full-length OmpX. (c) Thermal stability of spliced OmpX refolded into DPC micelles. (d) same as in (a) but with the α-helical membrane protein MPV17. (e) ESI-MS data of the intein fragments and the spliced MPV17 product. (f) Same as in (c) but with MPV17 in DPC micelles. (a,d) 0* indicates the ∼10 s time point right after mixing the two OmpX or MPV17 fragments.

Figure 3

Removal of ligation side products by insertion into lipid nanodiscs. (a) After intein splicing and refolding, properly folded full-length and a misfolded OmpX_C fragment are copurifying in DPC detergent micelles, giving rise to a 2D-[¹⁵N,¹H]-TROSY spectrum containing signals of both species (blue spectrum in panel b). The black spectrum in (b) is a reference with folded U-[²H,¹⁵N]-labeled OmpX in DPC micelles. Insertion into lipid nanodiscs selects for the properly folded species and efficiently removes the OmpX_C fragment, indicated by SDS-PAGE and a 2D-[¹⁵N,¹H]-TROSY spectrum lacking signals in the unfolded region (c). NMR spectra were recorded at 318 K and at 950 MHz ¹H frequency with a sample where the C-extein of OmpX (Figure 1b) is labeled with ²H and ¹⁵N (blue spectra). The N-extein is unlabeled and not visible in NMR. Trp sc: tryptophane NHε side chain signals.

Figure 4

2D-NMR analysis of the segmentally isotope-labeled integral membrane proteins OmpX and MPV17. (a) 2D-[¹⁵N,¹H]-TROSY spectra of uniformly labeled OmpX (gray) and segmentally labeled OmpX (red and blue). (b) Chemical shift perturbations (CSPs) within OmpX calculated from the spectra shown in (a). (c) CSPs mapped onto the structure of OmpX (2m06.pdb). The splicing site is indicated by the two green spheres. (d–f) Same as in (a–c) but with segmentally isotope labeled MPV17 in DPC micelles. The structural model of MPV17 was obtained with AlphaFold.

Figure 5

Segmental isotope labeling of membrane proteins facilitates NMR structure determination. (a) Structural interface between ²H,¹⁵N-labeled OmpX-N-extein and unlabeled OmpX-C-extein with observed NOE distance restraints (shown in (b)) within the labeled or to the unlabeled part. (c) and (d), same as in (a) and (b) but with an inverse isotope labeling pattern, i.e., ²H,¹⁵N-labeled OmpX-C-extein and unlabeled OmpX-N-extein. (e) 20 lowest energy structures obtained with the two segmentally labeled OmpX samples in lipid nanodiscs, showing an root mean square deviation (rmsd) of backbone atoms in ordered secondary structure elements of 0.29 Å. The assignment of NOE contacts was facilitated by the lower complexity and signal overlap in the individual spectra.