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Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR

Nature Protocols volume 13, pages 79–98 (2018)Cite this article

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Abstract

Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12–24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.

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Figure 1: Design of smaller nanodiscs for solution-state NMR spectroscopy.
Figure 2
Figure 3: Geometrical considerations on the available lipid bilayer surface area in a nanodisc that can be replaced by a target membrane protein.
Figure 4: Flow scheme for the optimization of nanodisc preparations for NMR spectroscopy.
Figure 5: Optimization of NMR spectra.
Figure 6: High-resolution structure determination of membrane proteins in smaller nanodiscs using multidimensional NMR spectroscopy and selective isotope labeling.
Figure 7: Measurement of residual dipolar couplings and paramagnetic relaxation enhancements of membrane proteins in phospholipid nanodiscs.

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Acknowledgements

We thank S. Hyberts (Harvard Medical School) for advice on NMR non-uniform sampling (NUS) setup and data processing. F.H. acknowledges support of the Technical University of Munich–Institute for Advanced Study, funded by the German Excellence Initiative and the European Union Seventh Framework Programme under grant agreement no. 291763, the Helmholtz Center Munich and the Helmholtz Society (grant VH-NG-1039), the Center for Integrated Protein Science Munich (CIPSM) and German Research Foundation (DFG) grant SFB1035 (project B13). M.L.N. acknowledges support from National Institutes of Health (NIH) grant F32GM113406. G.W. acknowledges support from the NIH through grants GM075879, GM047467, GM094608, AI037581 and EB002026.

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  1. Center for Integrated Protein Science Munich (CIPSM) at the Department of Chemistry and Institute for Advanced Study, Technical University of Munich, Garching, Germany

    Franz Hagn

  2. Helmholtz Center Munich, Institute of Structural Biology, Neuherberg, Germany

    Franz Hagn

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA

    Mahmoud L Nasr & Gerhard Wagner

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F.H. performed the experiments and analyzed the data; F.H. wrote the paper, and G.W. and M.L.N. commented on and edited the paper.

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Correspondence to Franz Hagn or Gerhard Wagner.

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Hagn, F., Nasr, M. & Wagner, G. Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13, 79–98 (2018). https://doi.org/10.1038/nprot.2017.094

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