Unfolding and identification of membrane proteins in situ

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 b...

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Bibliographic Details
Published in:eLife 2022-09, Vol.11
Main Authors: Galvanetto, Nicola, Ye, Zhongjie, Marchesi, Arin, Mortal, Simone, Maity, Sourav, Laio, Alessandro, Torre, Vincent
Format: Article
Language:English
Subjects:
Atomic force microscopy
Bayesian analysis
Lipid bilayers
Mass spectrometry
Mass spectroscopy
Mechanical properties
Membrane proteins
Membranes
Microscopy
protein identification
Proteins
Proteomics
Scientific imaging
single-molecule force spectroscopy
Structural Biology and Molecular Biophysics
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Summary: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.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.77427