by Christoph Weigel
Figure 1. Movie appr. in real time (i.e. depends also on your browser); actual rate without actin load ~100 rev/sec. Source.
Why on earth would one stare—even for a few seconds—at this blurry, low-res, pixelated movie (to the right), watching a jittery rod tumbling counterclockwise around its pivot? Because it's ATP synthase, a key enzyme of the energy metabolism in every living cell, that's rotating there. And when a molecule does its job by a rotational movement, well, then it takes a movie to demonstrate this. We turn thus to 'very small things considered': an enzymatically active ~530 kDa multi-subunit protein complex with a diameter of ~9 nm and a length of ~20 nm. Biophysicists finally showed in 1997 what biochemists and crystallographers had suspected for quite a while but could not prove by their own experimental methodologies: ATP synthase—also known as FoF1-ATPase, or complex V of the respiratory chain—is a molecular dynamo. First a closer look into the screenplay for the movie before peeking briefly at the bigger picture...
Molecular tinkering, the professional way
Noji and colleagues assembled their object of interest in three separate steps, all standard lab procedures, by 'affinity coupling' of the individual components. First, they prepared F1 particles (with the subunit composition: α3β3γ) of ATP synthase with His-tags at the N-termini of the β subunits to affix them to Ni-NTA covered microscope slides and a biotin-tag at the γ subunit. Second, to construct an actin probe conjugated to a red-orange fluorescent dye they coupled ~1 µm-rods of polymerized and biotinylated actin to rhodamine phalloidine. Lastly they coupled the biotinylated actin rods to the biotinylated γ subunits of ATP synthase via streptavidin. The whole assembly is shown schematically in Figure 2. When infused with ATP in buffer solution, a small percentage of the molecular assemblies started rotating―in all cases counterclockwise―as seen under the epifluorescence microscope and filmed for Figure 1.