All tubulins are not alike: Heterodimer dissociation differs among different biological sources

Alpha‐ and beta‐tubulin isotypes are differentially expressed during brain development

“…MTs consist of heterodimers of multiple alpha‐ and beta‐tubulins in a 1:1 stoichiometry (Montecinos‐Franjola, Chaturvedi, Schuck, & Sackett, 2019). They polymerize in a GTP‐dependent manner to form protofilaments, which further assemble into the functional microtubule, a hollow tube consisting of usually 13 protofilaments in mammals (Chaaban & Brouhard, 2017; Mitchison & Kirschner, 1984).…”

Quantifying the Monomer–Dimer Equilibrium of Tubulin with Mass Photometry

“… 5 , 6 , 7 , 8 , [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 To achieve the required sensitivity, many single-molecule studies have had to rely on labelling and although care was taken to ensure that tubulin was not damaged by labelling, a quick, simple, label-free method is desirable because it excludes any potential perturbations.…”

“… 8 The resulting values, measured here for porcine brain tubulin, closely match those reported for rat brain tubulin in the absence of GTP (2.8 nM) in recent analytical ultracentrifugation experiments. 10 By measuring dissociation of tubulin from different species and tissues, they report values ranging from 0.33 nM for chicken red blood cell tubulin to 47 nM for HeLa cell tubulin. This suggests that a of 3–10 nM may apply generally to mammalian brain tubulin.…”

Quantifying the monomer-dimer equilibrium of tubulin with mass photometry

“…[2,3,4] Studies on the thermodynamic and kinetic stability of αβ-tubulin heterodimers, which ultimately impact the assembly and stability of microtubules, have produced a broad range of binding affinities and kinetics, ranging over 5 orders of magnitude from 10 −11 to 10 −6 M, with dissociation rates from 10 −5 to 10 −2 s −1 . [5,6,7,8] These contradictory and inconsistent results can be ultimately attributed to a combination of factors including tissue source and sample preparation, [9] as well as the lack of experimental approaches capable of quantifying binding affinities in the sub-µM range in near-native conditions. Truly label-free approaches generally require µM concentrations or higher, with higher dilutions only accessible through labelling, surface-based methods, or a combination of both.…”

“…These results quantify the binding affinity for the tubulin heterodimer both in the apo and GTP bound state and provide an upper limit to the dissociation rate, which is orders of magnitude faster than reports based on surface plasmon resonance. [7] Although it has been shown that the K d of the tubulin dimer depends significantly on the protein's source, [9] due to the fact that tubulin is highly conserved one would expect the effect of GTP binding to be similar across different species. The reduction of the K d in the GTP bound state demonstrates the stabilising effect nucleotide has upon the dimer, which in light of the fast off rate suggests improved resistance to local fluctuations in tubulin concentration.…”