Dataset Information

Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

ABSTRACT: The ??-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 ??-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.

SUBMITTER: Fineberg A

PROVIDER: S-EPMC7763485 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

Fineberg Adam A Surrey Thomas T Kukura Philipp P

Journal of molecular biology 20201015 23

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 ...[more]

PMID: 33068635

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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

Dataset Information

Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

Publications

Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

Similar Datasets

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