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The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.

Titel:	Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.
Autor/in / Beteiligte Person:	Tapia-Rojo, R ; Mora, M ; Garcia-Manyes, S
Zeitschrift:	Nature protocols, Jg. 19 (2024-06-01), Heft 6, S. 1779-1806
Veröffentlichung:	London, UK : Nature Pub. Group, 2006-, 2024
Medientyp:	academicJournal
ISSN:	1750-2799 (electronic)
DOI:	10.1038/s41596-024-00965-5
Schlagwort:	Single Molecule Imaging methods Magnetics methods Nanotechnology methods Protein Conformation Protein Folding Proteins chemistry Proteins metabolism
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Review Language: English [Nat Protoc] 2024 Jun; Vol. 19 (6), pp. 1779-1806. <i>Date of Electronic Publication: </i>2024 Mar 11. MeSH Terms: Proteins* / chemistry ; Proteins* / metabolism ; Single Molecule Imaging / methods ; Magnetics / methods ; Nanotechnology / methods ; Protein Conformation ; Protein Folding References: Nano Lett. 2021 Apr 14;21(7):2953-2961. (PMID: 33765390) ; Nat Rev Mol Cell Biol. 2009 Jan;10(1):63-73. (PMID: 19197333) ; Biophys J. 1997 Apr;72(4):1541-55. (PMID: 9083660) ; Nat Rev Immunol. 2017 Nov;17(11):679-690. (PMID: 28757604) ; Biophys J. 2002 Jun;82(6):3314-29. (PMID: 12023254) ; Science. 1992 Nov 13;258(5085):1122-6. (PMID: 1439819) ; Nature. 2018 Sep;561(7723):401-405. (PMID: 30185907) ; Cell. 2005 Sep 9;122(5):683-92. (PMID: 16143101) ; Cold Spring Harb Protoc. 2012 Jan 01;2012(1):133-8. (PMID: 22194260) ; Nat Commun. 2016 Jun 27;7:12058. 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Single-molecule magnetic tweezers to probe the equilibrium dynamics of individual proteins at physiologically relevant forces and timescales.