Quantifying the Monomer–Dimer Equilibrium of Tubulin with Mass Photometry

Highlights

• Quantifying high affinity protein–protein interactions is experimentally difficult.

• We use mass photometry to determine $\alpha \beta$-tubulin heterodimer energetics and kinetics.

• The K_d of the dimer is 8.48 ± 1.22 nM in the absence of added GTP.

• This lowers to 3.69 ± 0.65 nM upon GTP addition with a k_off > 10⁻² s⁻¹.

• Mass photometry is uniquely suited to study protein–protein interactions.

Abstract

The $\alpha \beta$-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 $\alpha \beta$-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.