Effect of Tubulin Self-Association on GTP Hydrolysis and Nucleotide Exchange Reactions

23 March 2022, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


Tubulin nucleation, microtubule (MT) assembly, stability, and dynamics depend on GTP hydrolysis and nucleotide exchange reactions. We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied using HPLC for determining the concentrations of tubulin-free and tubulin-bound GTP and GDP and by SAXS and cryo-TEM. We observed no GTP hydrolysis below the critical conditions for MT assembly, despite the assembly of tubulin 1D curved oligomers and single rings, showing that their assembly did not involve GTP hydrolysis under our conditions. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to calculate, at each tubulin concentration, the distributions of single rings, 1D oligomers, and free dimers, and thereby the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites.


nucleotide exchange
GTP hydrolysis

Supplementary materials

Supporting Data and Analyses
The supporting information includes the following chapters: 1. Nucleotide Composition Variation of Different Sample Preparation Protocols 2. Variation in the HPLC Elution Profiles 3. Thermodynamic Model of Tubulin Self-Association into Single Rings and Curved 1D Oligomers 3.1 Standard Helmholtz Association Free Energies 4. Solving the Set of Equilibrium Reactions 4.1 Excluding the Difference Between Tubulin Dimers With Exposed and Buried E-Sites 4.2 Including the Difference Between Tubulin Dimers with Exposed and Buried E-sites 5. Complete Nucleotide and Tubulin Complexes Distributions 6. Nucleotide Content Bound to the Tubulin and Free in the Buffer at Steady-State 7. Mole Percent of GTP at Onset of the Hydrolysis Kinetics


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