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Cited by 13Force Microscopy Techniques and ApplicationsProtein Structure and DynamicsLipid Membrane Structure and Behavior

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Unfolding and identification of membrane proteins in situ

Open access
Galvanetto, Nicola, Ye, Zhongjie, Marchesi, Arin, Mortal, Simone, Maity, Sourav, Laio, Alessandro, Torre, Vincent·2022·eLife
DOI 10.7554/elife.77427PMC PMC9531951

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Abstract

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (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, which 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 four 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.

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Metadata

Authors
Galvanetto, N., Ye, Z., Marchesi, A., Mortal, S., Maity, S., Laio, A., & Torre, V.
Affiliations
  • Scuola Internazionale Superiore di Studi Avanzati (IT)
  • Kanazawa University (JP)
  • The Abdus Salam International Centre for Theoretical Physics (ICTP) (IT)
Corresponding author
Galvanetto, Nicola
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2022-09-12
Volume
11
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.77427
PMC
PMC9531951
OpenAlex
W4295292864
Cited by
13
References
1
Citations by year
2026: 3 · 2025: 4 · 2024: 3 · 2023: 2
FWCI
0.92
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, cc-by)
  • SDL (submittedVersion, cc-by)
  • Università Politecnica delle Marche (Università Politecnica delle Marche) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, cc-by)
Funders
H2020 European Research Council (MSCA IF-2014-EF-655157)
Topics
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Keywords
single-molecule force spectroscopy, plasma membrane isolation, force-distance curves, membrane proteomics
MeSH terms
Bayes Theorem, Lipid Bilayers, Membrane Proteins, Microscopy, Atomic Force, Proteomics, Protein Unfolding

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