It is possible to directly observe the rotation of the F1ATPase cam shaft using a fluorescence microscope, although considerable ingenuity is required. Noji et al (1997) Nature 386, 299-302 used a genetically modified F1ATPase from a thermophilic bacterium expressed in E. coli. They discarded the F0 basepiece and tethered the F1 motor head groups to a glass plate using polyhistidine tags attached to the N-termini of all three beta subunits. The glass plate had been pre-treated with horseradish peroxidase conjugated with the nickel complex of nitrilotriacetic acid, to which polyhistidine binds with high affinity. [Nitrilotriacetic acid looks like half an EDTA molecule, so it leaves un-coordinated nickel positions available for external ligands.]
The motors were glued down by their large catalytic subunits, leaving the motor shafts exposed, and facing away from the glass. The gamma subunits which form the shaft were modified by site directed mutagenesis to remove the original Cys193 (which is inconveniently far down the shaft) and replace it with serine. These workers also replaced Ser107 in the stalk region with cysteine.
This single cysteine residue (the only one in the molecule) could then be biotinylated, and linked using streptavidin to fluorescently labelled, biotinylated actin filaments. [Streptavidin has four biotin binding sites.] The fluorescent actin filaments were many times larger than the tethered motors and could be visualised in a light microscope. Addition of 2mM ATP caused a small number of motor shafts marked by the actin filaments to rotate in a counter-clockwise direction. The movie shows the results they obtained. Circular motion also occurs in the proteins which rotate bacterial flagella, another important enzyme system which is driven by the proton motive force. |
ATPase movieclick the arrow on the left to start the movie |