Q&A: Carol Robinson – Shedding light on a mysterious motor protein

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Prof. Carol Robinson

The enzyme ATPase has been described as the most efficient, beautiful machine that nature has ever made. Carol Robinson, a Royal Society Research Professor at the University of Oxford Department of Chemistry and a Wellcome Trust grantholder, has been using a new technique to find out more about this ‘intriguing beast’, as she calls it. She tells us about her latest study, the pinnacle of 15 years’ work.

Why is ATPase interesting?

It’s a fascinating rotary machine that is found in cells across all kingdoms of life. It’s made from about 30 protein ‘subunits’ that form two main parts: a motor that sits in the cell membrane and a head that protrudes into the cytoplasm [the liquid inside the cell]. The motor rotates a shaft that causes the head to spin.

ATPase has two important roles. As the head spins, it synthesises ATP [adenosine triphosphate, which transports energy within the cell]. It can also work in the other direction by consuming ATP and using the energy released to pump protons across the membrane, maintaining the acidity of cells. We were particularly interested in this second mode of action because it has a number of physiological roles and is associated with diseases such as kidney failure, osteoporosis and cancer.

When the head and the motor parts are together, they act in a coordinated way, but it’s known that these two parts separate in vivo, which has been proposed as a regulatory mechanism. When this happens, it’s likely that the head stops consuming ATP and the base stops pumping protons. One of many questions in the field has been: how do they stop when they become detached?

What were you hoping to find out?

Initially we were just curious whether ATPase could survive intact during electrospray mass spectrometry, which involves evaporating water droplets containing the protein. It was hard to imagine how a molecular motor, normally embedded in a cell membrane with water on either side, could survive under these conditions. We reasoned that if we could get it into a gas environment whilst still intact, we would probably learn something new about how the head and motor talk to each other.

What did you do?

To our surprise, we were able to evaporate whole ATPase assemblies [with both head and motor parts] without destroying them. We used a specially modified mass spectrometer that we’ve been optimising over the last 15 years, to separate protein assemblies according to their cross-section [which depends on protein mass, charge and shape]. Smaller, more mobile proteins travel through quickly, while big, lumbering ones take longer. It can tell you if a protein has changed shape, for example in response to binding a small molecule.

The technique was originally invented for individual protein molecules, so it was exciting to use it with these much larger membrane-embedded protein assemblies for the first time. We compared two different ATPases from different bacteria. We challenged them with various stimuli to see how they would respond, for example by changing the pH or ATP concentration.

What did you find out?

It had always been unclear whether lipid molecules [found in cell membranes] have a structural role in ATPase function. We were very surprised to find that lipids bind to the two ATPases in strikingly different ways, leading us to propose that they have different structural roles in regulating the motor proteins.

We also proposed a new explanation for how ATPase stops pumping protons when the head and motor parts separate. We saw that one of the protein subunits moved away from the proton channel [the hole through which protons are pumped], which made us think that other lipids might move in and shut off the channel. This would stop protons from leaking through.

What are you planning to do next?

We’re looking closely at lots of other membrane proteins. Understanding the role that drugs play in regulating them is the next major goal, which could have implications for lots of medical conditions as well as drug discovery programmes.

Reference

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