Fig. 1. The N-terminus of Op18 is sufficient to inhibit in vitro tubulin-tubulin interaction-dependent GTP hydrolysis. (A) Schematic representation of Op18 truncation derivatives. At the top, native Op18 is depicted with an unstructured N-terminus and two repeats of weakly homologous -helical regions according to previous reports (Gigant et al., 2000; Wallon et al., 2000). Phosphorylation sites are indicated with a `P' (Ser-16, Ser-25, Ser-38 and Ser-63). The positions of the two longitudinally arranged tubulin heterodimers along the two helical repeats are depicted according to the low resolution X-ray structure (Gigant et al., 2000), and the orientation of the N-terminus towards the -tubulin end of the tandem tubulin dimer complex is as suggested by cross-linking experiments (Muller et al., 2001). Each truncated Op18 derivative is denoted by the numbers within brackets that indicate the amino-acid residues present. The NR-helix corresponds to a 149 residue of a -helical portion from the rod region of non-muscle myosin heavy chain. (B) Tubulin (5 µM in PEM, pH 6.8) preloaded with -[³²P]-GTP was incubated at 37°C with increasing concentrations of each of the Op18 derivative outlined in A. Initial single-turnover hydrolysis rates were evaluated as described in Materials and Methods. Data are means of two independent experiments ±s.e.m. The experiment has also been performed using GST derivatives tagged at the N-terminus and the same result was produced (data not shown).