Looking at a single molecule

Our cells are full of all sorts of molecules: proteins, DNA, RNA and more. In this thick soup of interacting compounds, it is difficult to see what one particular molecule is doing. Antoine van Oijen, Professor of Single Molecule Biophysics, hopes to do just that. He has taken an important step towards achieving this goal by visualizing the movement in a single transport protein. His work will be published in the journal Nature on 3 October.

The molecule visualized by Van Oijen and his group is a transport protein that shuttles the nutrient aspartate across the membrane of microbial cells. It has the rather unpronounceable name of Glt_Ph, and belongs to a wider class of transport molecules in the cell membrane. ‘So far, no one has been able to visualize how the transport process through the membrane actually works’, Van Oijen explains.

Although the visualization of single molecules has been around for some 15 to 20 years, the technique hasn’t yet been applied to membrane proteins. ‘Just a handful of studies have been published on these proteins, but all in very artificial systems.’ It is rather tricky to experiment with proteins that are embedded in a cell membrane.

Transport proteins in the cell membrane are vital for the functioning of cells. They shuttle all sorts of compounds across the fatty membrane and maintain the gradient of ions such as sodium and potassium. This gradient in turn drives many transport proteins, much like how the flow of a river drives a waterwheel. Any malfunction in the transport system is dangerous and often lethal.

Van Oijen’s postdoc Guus Erkens managed to visualize the movement made by Glt_Ph, which is a complex of three identical subunits. Erkens labelled two of these with two different fluorescent markers. ‘I diluted both labels to a point where a reasonable amount of the complexes contained exactly one of each label.’

The first label emits light when excited by a laser. The second label catches this light and emits a different colour. ‘The amount of light it emits depends on the distance between these labels’, explains Erkens. This therefore makes it possible to see how far apart the two labelled units in the complex are.

Erkens measured the response of hundreds of lipid vesicles containing the labelled transport protein under a special microscope. He only accepted measurements from vesicles containing one properly labelled transporter for his analysis.

The main question Erkens wanted to answer was whether the three subunits work in unison or separately. ‘What we found out is that all three subunits work independently. They can each transport an aspartate molecule.’ The units move up and down through the membrane like a lift.

The research was carried out in close cooperation with Professor of Membrane Enzymology, Dirk Slotboom, who has spent ten years working with Glt_Ph. He recently published a paper in Nature Structural & Molecular Biology on Glt_Ph, see ‘The Mystery of the Empty Lift’.

Both studies have an important role in medical research, as the human brain has a similar transport complex for removing glutamate, a neurotransmitter that allows nerve cells to communicate with each other. ‘If this complex doesn’t work properly, neither does the brain’, explains Van Oijen. It is possible that a defect in glutamate transport has a role in conditions such as Alzheimer’s Disease, ALS and Huntington’s Disease.

‘Now that we know how the transporter is supposed to work, we will be able to search for abnormalities’, says Van Oijen. ‘This could help us develop more targeted drugs. However, the whole process could take another ten to twenty years.’

The main thrust of Van Oijen’s research is not developing new treatments, but rather understanding the multitude of processes going on in a cell. His focus is on individual molecules, which he aims to study in as natural an environment as possible.

‘Other research groups have studied Glt_Ph in a soapy solution. This allows it to flip over, but no transport takes place as the solution does not have an “inside” or “outside”.’ The results of this type of research are very different from Van Oijen’s latest results. He sees this as a sign that he is on the right track.

‘It is important to study these transport proteins in the right environment. We’ll only improve our understanding of the molecular mechanism if we can study real transport taking place through a real membrane.’ Van Oijen is now focusing on protein complexes consisting of multiple components. ‘Our ultimate aim is to find out how all these proteins work in a real cell.’

Reference: Unsynchronised subunit motion in single trimeric sodium-coupled aspartate transporters, Guus B. Erkens, Inga Hänelt, Joris M.H. Goudsmits, Dirk Jan Slotboom and Antoine M. van Oijen. Nature 3 oktober 2013, D OI:10.1038/nature12538