Quantifying the Monomer–Dimer Equilibrium of Tubulin with Mass Photometry

Surrey, Thomas; Kukura, Philipp

doi:10.1016/j.jmb.2020.10.013

Cited by 16 publications

(16 citation statements)

References 22 publications

(13 reference statements)

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“…A dissociation equilibrium constant, K d , of 0.01 nM was calculated from the measured dimer dissociation rate and the estimated dimer association rate . Recent mass photometry measurements reported K d of 3.7 nM . Hence, the expected contribution of tubulin monomers is indeed negligible (about 0.01%).…”

Section: Resultsmentioning

confidence: 99%

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Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association

et al. 2021

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Tubulin self-association is a critical process in microtubule dynamics. The early intermediate structures, energetics, and their regulation by fluxes of chemical energy, associated with guanosine triphosphate (GTP) hydrolysis, are poorly understood. We reconstituted an in vitro minimal model system, mimicking the key elements of the nontemplated tubulin assembly. To resolve the distribution of GTP- and guanosine diphosphate (GDP)-tubulin structures, at low temperatures (∼10 °C) and below the critical concentration for the microtubule assembly, we analyzed in-line size-exclusion chromatography–small-angle X-ray scattering (SEC-SAXS) chromatograms of GTP- and GDP-tubulin solutions. Both solutions rapidly attained steady state. The SEC-SAXS data were consistent with an isodesmic thermodynamic model of longitudinal tubulin self-association into 1D oligomers, terminated by the formation of tubulin single rings. The analysis showed that free dimers coexisted with tetramers and hexamers. Tubulin monomers and lateral association between dimers were not detected. The dimer–dimer longitudinal self-association standard Helmholtz free energies were −14.2 ± 0.4 k B T (−8.0 ± 0.2 kcal mol–1) and −13.1 ± 0.5 k B T (−7.4 ± 0.3 kcal mol–1) for GDP- and GTP-tubulin, respectively. We then determined the mass fractions of dimers, tetramers, and hexamers as a function of the total tubulin concentration. A small fraction of stable tubulin single rings, with a radius of 19.2 ± 0.2 nm, was detected in the GDP-tubulin solution. In the GTP-tubulin solution, this fraction was significantly lower. Cryo-TEM images and SEC-multiangle light-scattering analysis corroborated these findings. Our analyses provide an accurate structure–stability description of cold tubulin solutions.

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Section: Resultsmentioning

confidence: 99%

“…29 Recent mass photometry measurements reported K d of 3.7 nM. 30 Hence, the expected contribution of tubulin monomers is indeed negligible (about 0.01%).…”

Section: ■ Introductionmentioning

confidence: 99%

Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association

et al. 2021

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“…The αβ-heterodimer state is not fixed; rather, purified heterodimers can undergo reversible dissociation with moderately fast kinetics into stable monomers of α- and β-tubulin (Montecinos-Franjola et al, 2016). A wide range of dissociation constants have been reported for purified αβ-heterodimers, from 0.1 nM to 1.0 µM (Mejillano and Himes 1989; Detrich and Williams 1978; Caplow and Fee 2002; Fineberg et al, 2020). This apparent disagreement may be attributable to differences in experimental conditions but also to biologically relevant differences in tubulins.…”

Section: Introductionmentioning

confidence: 99%

Asymmetric requirement for α-tubulin over β-tubulin

Wethekam

Moore

2022

Preprint

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How cells regulate the supply of α- and β-tubulin monomers to meet the demand for αβ-heterodimers while avoiding consequences of monomer imbalance is not understood. We investigate the role of gene copy number in tubulin regulation and how shifting the expression of α- or β-tubulin genes impacts tubulin proteostasis and microtubule function. We find that α-tubulin gene copy number is important for maintaining an excess α-tubulin protein compared to β-tubulin protein and preventing accumulation of super-stoichiometric β-tubulin. Super-stoichiometric β-tubulin is toxic to cells, leading to loss of microtubules, formation of non-microtubule assemblies of tubulin, and disrupted cell proliferation. In contrast, decreased β-tubulin or increased α-tubulin has minor effects. We provide evidence that cells rapidly equilibrate the concentration of α-tubulin protein during shifts in α-tubulin isotype expression to maintain a ratio in excess of β-tubulin. We propose an asymmetric relationship between α- and β-tubulins, where α-tubulins are maintained in excess to supply αβ-heterodimers and limit the accumulation of β-tubulin monomers.

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“…A comparison by Wu et al of MP with established binding affinity measurement techniques showed that MP binding affinity data of antibody-antigen interactions was in agreement with ITC and BLI measurements and superior in terms of stoichiometry determination. 126 Recently, MP has also been used to quantify binding affinities in the self-association of proteins (FOXP2 oligomerization, 127 tubulin dimerization, 128 CaMKIIα oligomerization 129 ), and for qualitative assessment of affinities between serum haptoglobin protein and hemoglobin 130 and casposase and DNA. 131 Recently, Li et al and Olerinyova et al showed that MP methods can be extended to other classes of biomolecules, such as DNA 132 and membrane proteins.…”

Section: Solution-based Methodsmentioning

confidence: 99%