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.
Highlights
Microtubules, involved in processes as broad as mitosis, cell motility and intracellular transport, are constructed of heterodimers of a- and b- tubulin, highly conserved members of the tubulin/FtsZ family of proteins, with each subunit able to bind a GTP molecule.[1]
Our results demonstrate the capabilities of mass photometry for quantifying protein–protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation
Studies on the thermodynamic and kinetic stability of ab-tubulin heterodimers, which impacts the assembly and stability of microtubules, have produced a broad range of binding affinities and kinetics, ranging over 5 orders of magnitude from 10À11 to 10À6 M, with dissociation rates from 10À5 to 10À2 sÀ1.5–9 The variation in previous results can be attributed to a combination of various technical reasons and to biochemical differences between tubulins from different species.[10]
Summary
Microtubules, involved in processes as broad as mitosis, cell motility and intracellular transport, are constructed of heterodimers of a- and b- tubulin, highly conserved members of the tubulin/FtsZ family of proteins, with each subunit able to bind a GTP molecule.[1]. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the ab-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 >10À2 sÀ1.
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