Life – an intricate wind-up toy

“When I was your age, I played not with phones but with wind-up toys.”
“How does a wind-up toy work?”
“Just like how your body works!”

I still remember my obsession with a tiny pull-back toy car I had in my childhood. The more you drag the car back, the more difficult it gets, but the further it drives forward upon release. That was the best way to demonstrate your superior patience and arm length among your cousins, and you could enjoy the whole afternoon as long as your arm could drag.

Unfortunately, despite having dominated the mechanical toy world for almost a century, wind-up toys are now on the verge of extinction, due to the introduction of small, cheap alkaline batteries in the 1960s. Music boxes are perhaps one of the few surviving lineages taking their last breaths.

So how does a wind-up toy work? By energy conservation and conversion. In the simplest design, a spiral spring is attached to a winder and a gear. When you rotate the winder with force, the energy is stored in the wound up spring as potential energy. As you release the winder, the spring unfolds and this converts the stored energy into kinetic energy, driving the gear and in turn generating movement of the attached parts down the line.

And that is exactly what your body is constantly doing to keep you running.

Wind-up motor (image from ode.ca)

A wind-up motor for ATP

We now know that the molecule which provides us with the energy we need for the day is adenosine triphosphate, or ATP (no, not caffeine). Stored in chemical form, the energy can be released, via the breakdown of ATP into adenosine diphosphate (ADP), to fuel essentially all cellular processes that require energy, be it mechanical or enzymatic. To synthesize such a magical molecule, we need nothing but a nanoscale wind-up motor – ATP synthase, located in the powerhouse of the cell – mitochondria.

Not surprisingly, ATP synthases are among the most conserved protein complexes across all domains of life, as it provides the most fundamental need for all lifeforms (no, not alcohol). It is found in mitochondrial and chloroplast membranes, as well as in cytoplasmic membranes of bacteria. Even archaea, which are single-celled organisms in the third domain of life proclaimed to be the most ancient of all, use ATP synthase. One of the most studied forms is the FOF1-type ATPase, and of course, bright scientists have already figured out its crystal structure.

Bacterial FOF1 ATPase model

ATPase can rotate? Show me!

As its name suggests, the FOF1 ATPase contains two portions: soluble F1 (fraction 1), and membrane-bound FO (“F-Oh”, oligomycin-binding fraction). If you disassemble the ATPase and take a look at the soluble F1 portion (subunits α3β3γ), it looks exactly like a winder handle on a toy motor; alternatively, the subunits γc12 are equally similar to one. There is also an extra stalk structure (subunits ab2δ, also known as the stator) that appears to stabilize parts of the FOF1 ATPase. Indeed, for a winder to work it needs to rotate against a static structure. Then which subunits does the stator stabilise and which parts are allowed to rotate as the winder?

Which is the winder handle? (photo from sciplus.com)

In a series of studies since the early 1970s, Paul Boyer and others proposed that the γ subunit rotates approximately 100 times per second within the crown of the F1 portion (subunits α3β3) to produce ATP in the β subunits, which undergo conformational changes.

That was a compelling finding supported by meticulous work that later earned Boyer a Nobel prize in 1997, but scientists felt something was still missing. They wanted to see it by eye! If it is really rotating, we should be able to visualize something like the merry-go-round, right? How, though, could we visualize the rotation of a blob that is less than 10 nanometers long, wide and tall?

In March 1997, a Japanese group based in Yokohama came up and said, “Hold my beer, we got this.” They pasted the F1 ATPase (α3β3γ subunits) on a glass slide, and attached a fluorescent actin filament to the tip of the γ subunit1. Since it would be difficult to ask the incomplete ATPase to synthesize any ATP on a plain glass slide, they instead fed it with ATP. This is like removing all gears from a wind-up motor, and let the pre-wound spring unfold, while being attached to a flashing string. If it unfolds by rotation, so should the string. As predicted, the authors observed an impressive rotary action in the F1 ATPase of up to 4 revolutions per second as the enzyme consumed ATP, albeit with some fluctuations due to Brownian motion and occasional minor reversals at certain angles. This dramatic video captured under a fluorescence microscope was the first direct visualization of the rotary action of a single F1 ATPase protein complex.

F1 ATPase rotating with a fluorescent actin filament

Wait. Why is it only rotating 4 times per second by feeding on ATP, when it could rotate 25 times faster in the opposite direction (clockwise) while generating ATP as otherwise proposed? This is largely because the actin filament attached to the F1 ATPase was a long, heavy string. If the F1 ATPase were a cube box of around 1 meter in length, width and height, it would be rotating with a string 260 meters long with a diameter of 70 centimeters and 155 times heavier, completely spread out under water, 4 times per second. Given the enormous hydrodynamic resistance, the F1 ATPase is probably the most powerful rotary motor by size in biology.

Winding up the nanoscale motor

That was ATP consumption. What about ATP production? How can we prove that winding up the ATPase rotor in the opposite direction on a glass slide actually generates ATP? Together with his team in 2005, Hiroyuki Noji, a co-author in the first visualization study, elegantly attached a magnetic bead to the F1 ATPase and forced it to rotate clockwise using magnetic tweezers2. This time, the F1 ATPase generated ATP. How did they know? Because when they stopped the magnetic field after a while, the F1 ATPase started to automatically rotate anti-clockwise, a sign of ATP consumption, at a rate proportional to the detected ATP concentration. In addition, they showed that the efficiency of ATP synthesis was greatly enhanced by the presence of ε subunit, which was not previously considered as essential for the rotary action.

F1 ATPase made to generate ATP by magnetic tweezer

Our body certainly cannot generate nano-scale magnetic fields to wind up every FOF1 ATPase. Instead, they do it with electricity. As we eat, we digest the food and metabolize it, which accumulate reducing power in the mitochondria. This power is then funneled through the electron transport chain as electrons, during which positively-charged protons are pumped into the inter-membrane space, to build up a steep electrochemical gradient. Now powered with this gradient, the subunit a of the FO portion of ATPase carries protons down the gradient across the membrane. This pushes the subunit c to rotate clockwise together with the γδε subunits, while the α3β3 subunits of the F1 portion generate ATP as they remain static owing to the stabilizing stalk. As a result, electric energy built up in the mitochondria is converted to kinetic energy in the FOF1 ATPase wind-up motor, and then to chemical energy in ATP. And trillions of these tiny power plants run 24/7 in our body just so we wake up the next morning.

Mammalian FOF1 ATPase runs on electrochemical gradient (note the slight difference in subunit composition)

It seems like wind-up toys are far from extinct after all. Rather, biology has adopted it into the heart of its system millions of years ahead of our invention, and this legacy will likely live till the end of mankind. Whenever I walk into a gift shop and see a palm size music box, I would wind it up, let it play the boring Canon in D or Für Elise, and be grateful to the countless and tireless wind-up motors in my body.

References
1.    Nature. 386(6622):299-302 (1997)
2.    Nature. 433(7027):773-777 (2005)

Feature photo kindly provided by Natalie Von Der Lehr
ATPase drawings by me

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