Unfolding and identification of membrane proteins in situ.
atomic force microscopy
molecular biophysics
protein identification
rat
single-molecule force spectroscopy
structural biology
Journal
eLife
ISSN: 2050-084X
Titre abrégé: Elife
Pays: England
ID NLM: 101579614
Informations de publication
Date de publication:
12 09 2022
12 09 2022
Historique:
received:
28
01
2022
accepted:
08
09
2022
pubmed:
13
9
2022
medline:
7
10
2022
entrez:
12
9
2022
Statut:
epublish
Résumé
Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscopy (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.
Identifiants
pubmed: 36094473
doi: 10.7554/eLife.77427
pii: 77427
pmc: PMC9531951
doi:
pii:
Substances chimiques
Lipid Bilayers
0
Membrane Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2022, Galvanetto, Ye et al.
Déclaration de conflit d'intérêts
NG, ZY, AM, SM, SM, AL, VT No competing interests declared
Références
Nat Protoc. 2007;2(9):2191-7
pubmed: 17853875
Int J Endocrinol. 2018 Mar 20;2018:1505307
pubmed: 29755519
Biophys J. 2002 Dec;83(6):3578-88
pubmed: 12496125
Methods Mol Biol. 2018;1814:181-200
pubmed: 29956233
Structure. 2012 Dec 5;20(12):2185-90
pubmed: 23159125
Science. 2013 Nov 8;342(6159):741-3
pubmed: 24202172
Nat Commun. 2015 May 12;6:7093
pubmed: 25963832
Biochim Biophys Acta Biomembr. 2018 Dec;1860(12):2532-2538
pubmed: 30273580
J Mol Biol. 2006 Apr 21;358(1):255-69
pubmed: 16519899
J Proteomics. 2016 Mar 1;135:38-50
pubmed: 26768581
Sci Rep. 2017 Sep 20;7(1):12000
pubmed: 28931892
Pflugers Arch. 2008 Apr;456(1):101-15
pubmed: 18058125
Structure. 2012 Jan 11;20(1):121-7
pubmed: 22244761
Chem Soc Rev. 2012 Jul 21;41(14):4781-96
pubmed: 22648310
Physiol Rep. 2013 Nov;1(6):e00148
pubmed: 24400150
J Struct Biol. 2003 Jun;142(3):369-78
pubmed: 12781664
Nature. 2002 Oct 3;419(6906):520-6
pubmed: 12368866
J Proteome Res. 2008 Jul;7(7):2654-69
pubmed: 18489131
Int J Mol Sci. 2013 Jan 14;14(1):1589-607
pubmed: 23344058
J Neurochem. 2006 Aug;98(4):1126-40
pubmed: 16895580
Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):E3852-61
pubmed: 27317744
Structure. 2018 Jun 5;26(6):829-838.e4
pubmed: 29731231
ACS Nano. 2018 Mar 27;12(3):2719-2727
pubmed: 29390177
Science. 2014 Jun 27;344(6191):1492-6
pubmed: 24970081
Nature. 2021 Aug;596(7873):583-589
pubmed: 34265844
J Mol Biol. 2004 Jul 23;340(5):1143-52
pubmed: 15236973
Structure. 2016 Apr 5;24(4):585-594
pubmed: 27021164
Annu Rev Biophys. 2010;39:111-37
pubmed: 20192782
Pflugers Arch. 2021 Sep;473(9):1423-1435
pubmed: 34357442
Bioinformatics. 2007 Jan 15;23(2):e231-6
pubmed: 17237097
Methods Mol Biol. 2010;604:123-35
pubmed: 20013368
Mol Cell Proteomics. 2008 Jun;7(6):1053-66
pubmed: 18245078
J Am Chem Soc. 2017 Jul 26;139(29):9867-9875
pubmed: 28677396
Cancers (Basel). 2020 Jun 23;12(6):
pubmed: 32585958
Nature. 2002 Aug 29;418(6901):998-1002
pubmed: 12198551
Methods Mol Biol. 2018;1814:243-264
pubmed: 29956237
J Biol Chem. 2019 Feb 1;294(5):1602-1608
pubmed: 30541924
Cell Res. 2018 Jul;28(7):746-755
pubmed: 29700422
Biophys J. 2016 Nov 15;111(10):2086-2098
pubmed: 27851934
Nat Chem Biol. 2016 Nov;12(11):911-917
pubmed: 27595331
J Chem Phys. 2018 Mar 28;148(12):123335
pubmed: 29604819
Proc Natl Acad Sci U S A. 2015 May 19;112(20):E2715-24
pubmed: 25941368
Angew Chem Int Ed Engl. 2009;48(44):8306-8
pubmed: 19787673
J Biol Chem. 2007 Nov 16;282(46):33537-33544
pubmed: 17848582
Science. 2017 Mar 03;355(6328):945-950
pubmed: 28254940
Anal Chem. 2004 Jul 15;76(14):4193-201
pubmed: 15253663
Structure. 2013 Mar 5;21(3):426-37
pubmed: 23434406
Adv Mater. 2018 Oct;30(42):e1704680
pubmed: 30260506
Structure. 2017 Jul 5;25(7):1139-1144.e2
pubmed: 28625789
Nat Struct Mol Biol. 2015 Oct;22(10):795-802
pubmed: 26344570
Biophys J. 2007 Jan 1;92(1):225-33
pubmed: 17028145
Sci Rep. 2017 Sep 22;7(1):12140
pubmed: 28939866
Bioinformatics. 2020 Dec 22;36(20):5014-5020
pubmed: 32653898
Commun Biol. 2018 Apr 5;1:23
pubmed: 30271910
Proc Natl Acad Sci U S A. 1975 May;72(5):1758-62
pubmed: 1057168
Elife. 2020 Mar 09;9:
pubmed: 32149605
J Neurosci. 2007 Jul 11;27(28):7447-58
pubmed: 17626205
Nat Neurosci. 2019 May;22(5):691-699
pubmed: 30988527
Mol Cell Proteomics. 2005 Sep;4(9):1265-72
pubmed: 15958392
Science. 2000 Apr 7;288(5463):143-6
pubmed: 10753119
Annu Rev Biochem. 2008;77:127-48
pubmed: 18518819
Nat Commun. 2018 Sep 28;9(1):3978
pubmed: 30266906
Biophys J. 2018 Sep 4;115(5):757-762
pubmed: 30122292
Structure. 2005 Feb;13(2):235-42
pubmed: 15698567
Science. 2002 Jun 28;296(5577):2395-8
pubmed: 12089443
Structure. 2006 Mar;14(3):521-7
pubmed: 16531236
Proc Natl Acad Sci U S A. 2018 Mar 13;115(11):2658-2663
pubmed: 29483271
Nat Nanotechnol. 2012 Aug;7(8):525-9
pubmed: 22772862
J Mol Biol. 2008 Feb 8;376(1):35-41
pubmed: 18155728