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A saposin-lipoprotein nanoparticle system for membrane proteins

Nature Methods volume 13, pages 345351 (2016) | Download Citation

Abstract

A limiting factor in membrane protein research is the ability to solubilize and stabilize such proteins. Detergents are used most often for solubilizing membrane proteins, but they are associated with protein instability and poor compatibility with structural and biophysical studies. Here we present a saposin-lipoprotein nanoparticle system, Salipro, which allows for the reconstitution of membrane proteins in a lipid environment that is stabilized by a scaffold of saposin proteins. We demonstrate the applicability of the method on two purified membrane protein complexes as well as by the direct solubilization and nanoparticle incorporation of a viral membrane protein complex from the virus membrane. Our approach facilitated high-resolution structural studies of the bacterial peptide transporter PeptTSo2 by single-particle cryo-electron microscopy (cryo-EM) and allowed us to stabilize the HIV envelope glycoprotein in a functional state.

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Acknowledgements

This work was supported by the European Molecular Biology Organization (EMBO) (long-term fellowship to J.F.), the Swedish Research Council (grants 2014-5583 and 2013-3922 to P.N., grant 2013-4621 to H.G. and grant 2010-4483 to C.J.), the Knut and Alice Wallenberg Foundation (grant 2014.0112 to P.N.), the Swedish Cancer Society (grant 13 0401 to P.N. and grant 14 0473 to H.G.), the Swedish Childhood Cancer Foundation (grant PR2014-0156 to P.N.), Nanyang Technological University (start-up grant to P.N.), Marie Curie Actions Project FP7-People-ITN-2008 (Virus Entry 235649 to H.G.), Stiftelsen Läkare mot AIDS Forskningsfond (to R.L.), Howard Hughes Medical Institute (Y.C.), the US National Institutes of Health (grants R01GM098672, P50GM082250 and 1S10OD020054 to Y.C.), the Karolinska Institutet Center for Biosciences (to C.J.), the China Scholarship Council (fellowship to L.Z.) and the Molecular Medicine Partnership Unit (MMPU) of the University Clinic Heidelberg (A.F.-P.S. and J.A.G.B.) and the European Molecular Biology Laboratory (A.F.-P.S. and J.A.G.B.). We also acknowledge the Protein Science Facility at Karolinska Institutet for cloning assistance and protein purification. Y.C. is an investigator at Howard Hughes Medical Institute.

Author information

Author notes

    • Jens Frauenfeld

    Present address: Salipro Biotech AB, Södertälje, Sweden.

Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Jens Frauenfeld
    • , Fatma Guettou
    • , Per Moberg
    • , Christian Löw
    •  & Pär Nordlund
  2. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

    • Robin Löving
    •  & Henrik Garoff
  3. Keck Advanced Microscopy Laboratory, Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA.

    • Jean-Paul Armache
    •  & Yifan Cheng
  4. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

    • Andreas F-P Sonnen
    •  & John A G Briggs
  5. Molecular Medicine Partnership Unit, European Molecular Biology Laboratory–Universitätsklinikum Heidelberg, Heidelberg, Germany.

    • Andreas F-P Sonnen
    •  & John A G Briggs
  6. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden.

    • Lin Zhu
    •  & Caroline Jegerschöld
  7. School of Technology and Health, Royal Institute of Technology, Novum, Huddinge, Sweden.

    • Lin Zhu
    •  & Caroline Jegerschöld
  8. EMBL Hamburg, Hamburg, Germany.

    • Ali Flayhan
    •  & Christian Löw
  9. Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA.

    • Yifan Cheng

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Contributions

J.F. developed the concept, performed experiments, analyzed data and wrote the manuscript. R.L. developed, performed and analyzed HIV-related experiments. J.-P.A. carried out all cryo-EM experiments, including data acquisition, processing and data interpretation. F.G., P.M., A.F. and C.L. purified membrane proteins. A.F.-P.S. prepared grids, collected negative-stain data and conducted image processing on Salipro-T2. C.J. and L.Z. prepared grids and collected negative-stain data on lipid-only Salipro nanoparticles. J.A.G.B., H.G., Y.C. and P.N. contributed project feedback and comments on the manuscript. All authors contributed to data interpretation and preparation of the manuscript.

Competing interests

Some of the authors have filed patent applications related to this work (J.F.: EP 2 745 834, WO 2014/095576; J.F., R.L. and H.G.: WO 2015/036549). J.F. and R.L. are shareholders of Salipro Biotech AB.

Corresponding authors

Correspondence to Jens Frauenfeld or Pär Nordlund.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Table 1

Videos

  1. 1.

    Cryo-EM structure of Salipro-POT.

    3D density map of the tetrameric bacterial transporter with each of the subunits and tentative placement of Saposin (orange and blue, respectively) in the density.

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DOI

https://doi.org/10.1038/nmeth.3801

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