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Quantifying the monomer-dimer equilibrium of tubulin with mass photometry

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dc.contributor.author Fineberg, Adam
dc.contributor.author Surrey, Thomas
dc.contributor.author Kukura, Philipp
dc.date.accessioned 2020-11-23T07:04:41Z
dc.date.available 2020-11-23T07:04:41Z
dc.date.issued 2020
dc.identifier.citation Fineberg A, Surrey T, Kukura P. Quantifying the monomer-dimer equilibrium of tubulin with mass photometry. J Mol Biol. 2020 Nov 20;432(23):6168-72. DOI: 10.1016/j.jmb.2020.10.013
dc.identifier.issn 0022-2836
dc.identifier.uri http://hdl.handle.net/10230/45847
dc.description.abstract The αβ-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <µM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αβ-tubulin dissociation constant (8.48±1.22 nM) and its tightening in the presence of GTP (3.69±0.65 nM), at a dissociation rate >10-2 s-1. Our results demonstrate the capabilities of mass photometry for quantifying protein-protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation.
dc.description.sponsorship We thank the Surrey lab members, The Francis Crick Institute, for tubulin purification including Nicholas Cade for his assistance in early experiments. Research in the Kukura group is supported by the ERC (CoG 819593), TS was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163), the UK Medical Research Council (FC001163), and the Wellcome Trust (FC001163). T.S. also acknowledges support from the European Research Council (Advanced Grant, project 323042) and from the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme of the Generalitat de Catalunya.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.publisher Elsevier
dc.relation.ispartof J Mol Biol. 2020 Nov 20;432(23):6168-72
dc.rights © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article distributed under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
dc.rights.uri http://creativecommons.org/licenses/by/4.0/
dc.title Quantifying the monomer-dimer equilibrium of tubulin with mass photometry
dc.type info:eu-repo/semantics/article
dc.identifier.doi http://dx.doi.org/10.1016/j.jmb.2020.10.013
dc.subject.keyword Binding affinity
dc.subject.keyword Mass photometry
dc.subject.keyword Single molecule
dc.subject.keyword Tubulin
dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/819593
dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/323042
dc.rights.accessRights info:eu-repo/semantics/openAccess
dc.type.version info:eu-repo/semantics/publishedVersion

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