Unfolding and identification of membrane proteins in situ

  1. Nicola Galvanetto  Is a corresponding author
  2. Zhongjie Ye
  3. Arin Marchesi
  4. Simone Mortal
  5. Sourav Maity
  6. Alessandro Laio
  7. Vincent Aldo Torre
  1. International School for Advanced Studies, Italy
  2. Kanazawa Medical University, Japan
  3. University of Groningen, Netherlands

Abstract

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an AFM to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, that involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from Mass Spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing 4 constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment.

Data availability

Data not present as Supplementary data are available in https://github.com/galvanetto/NativeSMFS/releases as well as the open source software to read them.

The following data sets were generated
    1. Galvanetto N
    2. Ye Z
    (2022) Data from native SMFS
    Github, https://github.com/galvanetto/NativeSMFS/releases.

Article and author information

Author details

  1. Nicola Galvanetto

    International School for Advanced Studies, Trieste, Italy
    For correspondence
    n.galvanetto@bioc.uzh.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0408-1747
  2. Zhongjie Ye

    International School for Advanced Studies, Trieste, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0306-5267
  3. Arin Marchesi

    Nano Life Science Institute, Kanazawa Medical University, Kanazawa, Japan
    Competing interests
    The authors declare that no competing interests exist.
  4. Simone Mortal

    International School for Advanced Studies, Trieste, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6534-9324
  5. Sourav Maity

    Moleculaire Biofysica, University of Groningen, Groningen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  6. Alessandro Laio

    International School for Advanced Studies, Trieste, Italy
    Competing interests
    The authors declare that no competing interests exist.
  7. Vincent Aldo Torre

    International School for Advanced Studies, Trieste, Italy
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8133-3584

Funding

H2020 European Research Council (MSCA IF-2014-EF-655157)

  • Arin Marchesi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Patricia Bassereau, Institut Curie, France

Version history

  1. Preprint posted: August 13, 2019 (view preprint)
  2. Received: January 28, 2022
  3. Accepted: September 8, 2022
  4. Accepted Manuscript published: September 12, 2022 (version 1)
  5. Version of Record published: October 4, 2022 (version 2)
  6. Version of Record updated: October 7, 2022 (version 3)

Copyright

© 2022, Galvanetto et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,656
    views
  • 401
    downloads
  • 4
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nicola Galvanetto
  2. Zhongjie Ye
  3. Arin Marchesi
  4. Simone Mortal
  5. Sourav Maity
  6. Alessandro Laio
  7. Vincent Aldo Torre
(2022)
Unfolding and identification of membrane proteins in situ
eLife 11:e77427.
https://doi.org/10.7554/eLife.77427

Share this article

https://doi.org/10.7554/eLife.77427

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics
    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article Updated

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.