Molecular dissection of MT binding by Bim1p. (A) Cartoons of Bim1 variants (WT or mutants lacking the CH, flexible linker, or dimerization domain) expressed as EGFP fusion proteins in E. coli. (B) Coomassie-stained gel of the respective Bim1 variants. (C) 1 µM of purified Bim1p constructs was subjected to MT binding and centrifuged. The amount of Bim1p bound to MTs was measured fluorometrically, and averaged data from three experiments were plotted as binding affinity curves. The graph compares the binding affinities of dimeric versus monomeric Bim1p variants. (D) The binding affinities of different dimeric Bimp1 constructs are compared. Kd and Bmax values for the different constructs are summarized in the table. (E) Light-scattering experiment showing the effects of Bim1 variants on the kinetics of tubulin polymerization. (C–E) Error bars denote standard error of the mean.
