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Abstract
Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.
Highlights
Microtubule (MT)1 filaments are dynamic polymers made of ␣/-tubulin heterodimers that are essential for a wide variety of central cellular functions in all eucaryotes, including cell transport, cell motility, and mitosis
At 10 °C, the reaction is endothermic, and the reaction heats decrease with increasing injection numbers as less and less free tubulin is available for stathmin binding
Mechanism of Tubulin-Stathmin Complex Formation—The binding equilibrium between tubulin and stathmin has been investigated by surface plasmon resonance [17, 19, 22, 32], by pull-down experiments [26, 28, 41], and most recently by a non-equilibrium-perturbing sequestration assay [31]
Summary
Protein Preparations—The cloning of the human full-length stathmin cDNA into the bacterial expression vector pET-16b (Novagen) is outlined in Ref. 13. The sample cell (volume 1.4 ml) was filled with a ϳ10 M tubulin solution in G buffer pH 6.8 or 7.5 supplemented with either 1 mM GTP or 2 mM GDP. During the course of the titration the total tubulin concentration is slightly diluted due to the addition of stathmin This effect is small but was taken into account in the evaluation. Samples of 2H/13C/15N-labeled stathmin and unlabeled tubulin were in G buffer at pH 6.8 or 7.5 Both 15N-1H HSQC and 15N-1H TROSY experiments were recorded. Analysis of the Tubulin-Stathmin Binding Equilibrium concentration in the calorimeter cell varies during the course of the titration and increases from 0.3 M after the first injection to 10.5 M after 40 injections. The measurement was carried out at 10 °C in 80 mM PIPES-KOH, pH 6.8, 1 mM MgCl2, 1 mM EGTA, 1 mM GTP
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