Motility of dimeric ncd on a metal-chelating surfactant: Evidence that ncd is not processive

The surface immobilization methods that allowed single-molecule motility experiments with native kinesin have not worked with the ncd motor protein and other kinesin-related motors. To solve this problem, a surfactant (Pluronic F108) was chemically modified with the metal-chelating group nitrilotriacetic acid (NTA) to allow surface immobilization of histidine-tagged microtubule motors. The chelating surfactant provided a convenient and effective method for immobilization and subsequent motility experiments with a dimeric H-tagged ncd protein (H-N195). In experiments with the absorption of H-N195 to polystyrene (PS) beads coated with F108-NTA, a monolayer of H-N195 bound in the presence of Ni2+, while in the absence of Ni2+, the extent of adsorption of H-N 195 to PS beads was greatly reduced. Zn motility experiments with H-N195 immobilized on F108-NTA-coated surfaces, microtubules moved smoothly and consistently at an average speed of 0.16 +/- 0.01 mu m/s in the presence of Ni2+, while without Ni2+, no microtubules landed on the F108-NTA-coated surfaces. Investigation of H-N195 motility on the F108-NTA surfaces provided several indications that ncd, unlike kinesin, is not processive. First, a critical H-N195 surface density for microtubule motility of approximately 250 molecules/mu m(2) was observed. Second, microtubule landing rates as a function of H-N195 surface density in che presence of MgATP suggested that several H-N195 molecules must cooperate in microtubule landing. Third, the ATP KM in motility assays (235 mu M) was substantially higher than the ATP KM Of dimeric ncd in solution (23 mu M) [Foster, K. A., Correia, J. J., and Gilbert, S. P. (1998) J. Biol. Chem. 273, 35307-35318].