Abstract
Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.
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Acknowledgements
This work was partially supported by NIH R01GM121725 to V.A.F.; Spanish Ministry of Science, Innovation and Universities and FEDER grant BFU2015-70552-P to V.A.F. and A.V.S.; Russian Science Foundation grant 17-75-30064 to P.V.B.; and the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government and the Ministry of Science and Higher Education of the Russian Federation.
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Conceptualization, V.A.F., P.V.B., A.V.S.; methodology, V.A.F., P.V.B.; investigation, K.C., P.A., J.V.L., P.V.B.; theoretical analysis, V.A.F., P.I.K., P.V.B.; writing of original draft, V.A.F., P.V.B., A.V.S.; review and editing, all authors; funding acquisition, V.A.F., P.V.B., A.V.S.; resources, V.A.F., P.V.B., A.V.S.; supervision, V.A.F., P.V.B., A.V.S.
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Peer review information Nature Protocols thanks Patricia Bassereau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Frolov, V. A. et al. Proc. Natl Acad. Sci. USA 100, 8698–8703 (2003): https://www.pnas.org/content/100/15/8698
Bashkirov, P. V. et al. Cell 135, 1276–1286 (2008): https://www.cell.com/cell/fulltext/S0092-8674(08)01503-1
Shnyrova, A. V. et al. Science. 339, 1433–1436 (2013): https://science.sciencemag.org/content/339/6126/1433.full
Mattila, J.-P. et al. Nature 524, 109–113 (2015): https://www.nature.com/articles/nature14509
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Source data (two columns x and y axes).
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Statistical source data (two columns for gray and black histograms).
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Statistical source data (for the histograms in Fig. 10a and b).
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Bashkirov, P.V., Kuzmin, P.I., Chekashkina, K. et al. Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes. Nat Protoc 15, 2443–2469 (2020). https://doi.org/10.1038/s41596-020-0337-1
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DOI: https://doi.org/10.1038/s41596-020-0337-1
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