Unfolding and identification of membrane proteins in situ
- International School for Advanced Studies, Italy
- Kanazawa Medical University, Japan
- 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.
-
Data from native SMFSGithub, https://github.com/galvanetto/NativeSMFS/releases.
Article and author information
Author details
Vincent Aldo Torre
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
- Patricia Bassereau, Institut Curie, France
Version history
- Preprint posted: August 13, 2019 (view preprint)
- Received: January 28, 2022
- Accepted: September 8, 2022
- Accepted Manuscript published: September 12, 2022 (version 1)
- Version of Record published: October 4, 2022 (version 2)
- 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
-
- 1,661
- views
-
- 403
- 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)
Unfolding and identification of membrane proteins in situ
eLife 11:e77427.
https://doi.org/10.7554/eLife.77427
Further reading
-
- Structural Biology and Molecular Biophysics
The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
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.
