The effect of various nucleotides and E491A mutation on equilibrium binding of MCAK to tubulin dimers. (A) Tubulin-binding assays with 700 nM MCAK, 150 μM of indicated nucleotide, and bound versus unbound bovine brain tubulin. (B) Dissociation constants (K_d) from fit curves. (C) Comparison of tubulin retention by wt MCAK and E491A in various nucleotides (1.6 mM) and a constant concentration of tubulin (2 μM). The percentage of tubulin dimer bound to the motor in each nucleotide and standard deviation from the mean (SD) are indicated. All lanes, respectively, are run on the same gel but the order of the lanes in E491A (bottom) are arranged to correspond to the nucleotide order of wt MCAK (top).