Tubulin Heterodimers Reversibly Dissociate with Moderate Kinetics as Demonstrated using Sedimentation Velocity Analytical Ultracentrifugation

Abstract

A number of studies have reported reversible αβ-tubulin heterodimer dissociation with Kd =10−8 - 10−7 M, with presumably rapid but undetermined kinetics. However, in a recent study an extremely tight association between subunits (Kd 3 hours). In this work we have revisited the thermodynamics and kinetics of rat brain αβ-tubulin (RBT) dissociation by taking advantage of sedimentation velocity analytical ultracentrifugation methodologies (SV-AUC) developed in recent years. In particular, a combination of optical detection systems was used to cover a wide range of protein concentrations: pseudo-absorbance optics (pABS-AUC) for micro- and submicromolar concentrations, and the newly developed fluorescence optics (FDS-AUC) for nano- and subnanomolar concentrations. SV-AUC data was modeled using SEDFIT software resulting in a sedimentation coefficient continuous distribution, c(s), from which the weight average sedimentation coefficients (sw) were obtained. The isotherms obtained from calculated sw as a function of tubulin concentration were analyzed in SEDPHAT software using the heteroassociation model A+B1 AB to compute the limiting sedimentation coefficients of the dimer and monomer species as well as the equilibrium dissociation constant. In our experimental conditions, RBT heterodimer sediments with sw = 5.1 s, while the monomer species co-sediments with sw = 2.9 s. According to the heteroassociation model, the RBT heterodimer dissociates with Kd = 5.5 x 10−8 M. From the analysis of sedimentation profiles we find the characteristic dissociation time constant to be on the order of hours. In conclusion, the αβ-tubulin heterodimer displays reversible dissociation, moderate Kd and moderate dissociation kinetics.