Motility of Dimeric Ncd on a Metal-Chelating Surfactant: Evidence That Ncd Is Not Processive

Abstract

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-N195 to PS beads was greatly reduced. In 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 μm/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/μm² was observed. Second, microtubule landing rates as a function of H-N195 surface density in the presence of MgATP suggested that several H-N195 molecules must cooperate in microtubule landing. Third, the ATP K_M in motility assays (235 μM) was substantially higher than the ATP K_M of dimeric ncd in solution (23 μM) [Foster, K. A., Correia, J. J., and Gilbert, S. P. (1998) J. Biol. Chem. 273, 35307−35318].