FIGURE 6:

αβ-Tubulin binds less tightly to, and undergoes catastrophe less frequently with, 2′-deoxy guanine nucleotides. (A) The 2′ hydroxyl of GTP, which is absent in dGTP, makes a hydrogen bond with a conserved asparagine (N202). PDB 4I4T (Prota et al., 2013) was used for this illustration. (B) Affinities of yeast αβ-tubulin for GDP and dGDP measured using a fluorescence-based competition assay (see Materials and Methods). αβ-Tubulin binds to dGDP about twofold less tightly than to GDP. Each data point represents the mean fraction of signal restored measured at a particular concentration of GDP or dGDP (n = 6 for GDP, blue; n = 4 for dGDP, purple; error bars represent SEM). (C) MTs polymerizing with dGTP show comparable concentration-dependent elongation rates to those growing with GTP MTs (four MTs per data point; error bars show SD). Parameters slope and y-intercept from the linear regression for elongation in the presence of dGTP are, respectively, 15.8 ± 0.06 μM⁻¹ s⁻¹ and −0.5 ± 0.02 s⁻¹. (D) MTs assembled with dGTP undergo catastrophe approximately fourfold less frequently than with GTP. Inset, plot of catastrophe frequency vs. MT growth velocity showing that the small increase in growth rate cannot explain the change in catastrophe frequency. Each data point represents the catastrophe frequency measured at a particular concentration of αβ-tubulin From left to right, for wild type + GTP (blue), n = 10, 21, 75, and 75; and for wild type + dGTP (purple), n = 43, 23, and 18. Error bars represent SD.