Quantifying the Monomer–Dimer Equilibrium of Tubulin with Mass Photometry

The [Formula: see text]-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 [Formula: see text]-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.