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.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.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.doi http://dx.doi.org/10.1016/j.jmb.2020.10.013
• dc.identifier.issn 0022-2836
• dc.identifier.uri http://hdl.handle.net/10230/45847
• dc.language.iso eng
• dc.publisher Elsevier
• dc.relation.ispartof J Mol Biol. 2020 Nov 20;432(23):6168-72
• dc.relation.projectID info:eu-repo/grantAgreement/EC/H2020/819593
• dc.relation.projectID info:eu-repo/grantAgreement/EC/FP7/323042
• 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.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by/4.0/
• dc.subject.keyword Binding affinity
• dc.subject.keyword Mass photometry
• dc.subject.keyword Single molecule
• dc.subject.keyword Tubulin
• dc.title Quantifying the monomer-dimer equilibrium of tubulin with mass photometry
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion