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Stabilization and structure determination of integral membrane proteins by termini restraining

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Abstract

Integral membrane proteins isolated from cellular environment often lose activity and native conformation required for functional analyses and structural studies. Even in their native state, they lack sufficient surfaces to form crystal contacts. Furthermore, most of them are too small for cryogenic electron microscopy detection and too big for solution NMR. To overcome these difficulties, we recently developed a strategy to stabilize the folded state of membrane proteins by restraining their two termini with a self-assembling protein coupler. The termini-restrained membrane proteins from distinct functional families retain their activities and show increased stability and yield. This strategy enables their structure determination at near-atomic resolution by facilitating the entire pipeline from crystallization, crystal identification, diffraction enhancement and phase determination, to electron density improvement. Furthermore, stabilization of membrane proteins enables their biochemical and biophysical characterization. Here we present the protocol of membrane protein engineering (2 weeks), quality assessment (1–2 weeks), protein production (1–6 weeks), crystallization (1–2 weeks), diffraction improvement (1–3 months) and crystallographic data analysis (1 week). This protocol is intended not only for structural biologists, but also for biochemists, biophysicists and pharmaceutical scientists whose research focuses on membrane proteins.

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Fig. 1: Overview of the concept and protocol of termini restraining.
Fig. 2: Quality assessment of restrained membrane proteins.
Fig. 3: Crystal identification and diffraction improvement of restrained membrane protein.
Fig. 4: Structure determination using the coupler structure and diffraction data.
Fig. 5: Expression vectors and construct generation.

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Data availability

The data presented in this protocol have been previously published13,14,15,16,17, and associated raw data are provided in the original articles. Source data are provided with this paper.

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Acknowledgements

This work is supported by the W. M. Keck Foundation (Forefront of Science Award), NHLBI (R01 HL121718), Children’s Discovery Institute (MCII 2020-854), NEI (R21 EY028705), NIGMS (R01 GM131008) and American Heart Association (20CSA35310354) to W.L. Crystallographic data collection used NE-CAT beamlines (GM124165), a Pilatus detector (RR029205) and an Eiger detector.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    Shixuan Liu, Shuang Li, Andrzej M. Krezel & Weikai Li

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  1. Shixuan Liu
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  2. Shuang Li
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  3. Andrzej M. Krezel
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  4. Weikai Li
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Contributions

W.L., S. Liu and S. Li conceived the study. S. Liu, S. Li and W.L. developed the protocols. W.L. and S. Liu wrote the manuscript with contributions from S. Li and A.M.K.

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Correspondence to Weikai Li.

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Peer review information Nature Protocols thanks Bernadette Byrne and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Liu, S. et al. Sci. Adv. 6, eabe3717 (2020): https://doi.org/10.1126/sciadv.abe3717

Liu, S. et al. Science 371, eabc5667 (2021): https://doi.org/10.1126/science.abc5667

Yang, Y. et al. EMBO J. 39, e105246, (2020): https://doi.org/10.15252/embj.2020105246

Supplementary information

Supplementary Information

Supplementary Methods, Supplementary Table 1 and Supplementary Fig. 1.

Supplementary Data 1

DNA sequence of sfGFP in pPICZ-derivatized vector

Supplementary Data 2

DNA sequence of sfGFP restrained human VKOR in pPICZ-derivatized vector

Supplementary Data 3

DNA sequence of sfGFP restrained DsbB in PET28b vector

Source data

Source Data Fig. 2

Unprocessed western blots.

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Liu, S., Li, S., Krezel, A.M. et al. Stabilization and structure determination of integral membrane proteins by termini restraining. Nat Protoc 17, 540–565 (2022). https://doi.org/10.1038/s41596-021-00656-5

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