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

The surface immobilization methods that allowed single-molecule motility ex periments with native kinesin have not worked with the ncd motor protein an d other kinesin-related motors. To solve this problem, a surfactant (Pluron ic F108) was chemically modified with the metal-chelating group nitrilotria cetic acid (NTA) to allow surface immobilization of histidine-tagged microt ubule motors. The chelating surfactant provided a convenient and effective method for immobilization and subsequent motility experiments with a dimeri c H-tagged ncd protein (H-N195). In experiments with the absorption of H-N1 95 to polystyrene (PS) beads coated with F108-NTA, a monolayer of H-N195 bo und in the presence of Ni2+, while in the absence of Ni2+, the extent of ad sorption of H-N 195 to PS beads was greatly reduced. Zn motility experiment s with H-N195 immobilized on F108-NTA-coated surfaces, microtubules moved s moothly 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-N TA-coated surfaces. Investigation of H-N195 motility on the F108-NTA surfac es provided several indications that ncd, unlike kinesin, is not processive . First, a critical H-N195 surface density for microtubule motility of appr oximately 250 molecules/mu m(2) was observed. Second, microtubule landing r ates as a function of H-N195 surface density in che presence of MgATP sugge sted that several H-N195 molecules must cooperate in microtubule landing. T hird, the ATP KM in motility assays (235 mu M) was substantially higher tha n 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].