deCastro M J, Ho C H, Stewart R J
Department of Bioengineering, University of Utah, Salt Lake City 84112, USA.
Biochemistry. 1999 Apr 20;38(16):5076-81. doi: 10.1021/bi9829175.
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].
能够用于天然驱动蛋白单分子运动实验的表面固定方法，对ncd运动蛋白和其他驱动蛋白相关的马达蛋白却不起作用。为了解决这个问题，一种表面活性剂（普朗尼克F108）用金属螯合基团次氮基三乙酸（NTA）进行了化学修饰，以实现对组氨酸标记的微管马达的表面固定。这种螯合表面活性剂为用二聚体H标记的ncd蛋白（H-N195）进行固定及后续运动实验提供了一种便捷有效的方法。在用H-N195吸附到涂有F108-NTA的聚苯乙烯（PS）珠的实验中，在存在Ni2+的情况下，H-N195形成了单层结合，而在不存在Ni2+时，H-N195对PS珠的吸附程度大大降低。在用固定在涂有F108-NTA的表面上的H-N195进行的运动实验中，在存在Ni2+的情况下，微管以0.16±0.01微米/秒的平均速度平稳且持续地移动，而在没有Ni2+时，没有微管落在涂有F108-NTA的表面上。对H-N195在F108-NTA表面上的运动研究提供了几个迹象，表明与驱动蛋白不同，ncd不是持续运动的。首先，观察到微管运动所需的关键H-N195表面密度约为250个分子/微米²。其次，在存在MgATP的情况下，微管着陆率作为H-N195表面密度的函数表明，几个H-N195分子必须协同作用才能使微管着陆。第三，运动测定中的ATP KM（235微摩尔）大大高于溶液中二聚体ncd的ATP KM（23微摩尔）[福斯特，K.A.，科雷亚，J.J.，和吉尔伯特，S.P.（1998年）《生物化学杂志》273，35307 - 35318]。
