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-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 micrometer/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/micrometer(2) 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 KM in motility assays (235 microM) was substantially higher than the ATP KM of dimeric ncd in solution (23 microM) [Foster, K. A., Correia, J. J., and Gilbert, S. P. (1998) J. Biol. Chem. 273, 35307-35318].
deCastro
M J
MJ
Department of Bioengineering, University of Utah, Salt Lake City 84112, USA.
Ho
C H
CH
Stewart
R J
RJ
D000255:Adenosine Triphosphate / Q000502:physiology; D002614:Chelating Agents / Q000737:chemistry*; D019281:Dimerization; D029721:Drosophila Proteins; D004800:Enzymes, Immobilized / Q000737:chemistry; D006639:Histidine / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D009038:Motion; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry*
D002614:Chelating Agents; D029721:Drosophila Proteins; D004800:Enzymes, Immobilized; C118682:F108-NTA, pluronic; D011994:Recombinant Proteins; D013501:Surface-Active Agents; C066696:ncd protein, Drosophila; D006639:Histidine; D000255:Adenosine Triphosphate; D016547:Kinesins; D009571:Nitrilotriacetic Acid