New technique to monitor protein folding (article by Leiden researchers Martin van Son and Martina Huber and others)


Biophysicists at the Leiden Institute of Physics, under supervision of Prof. Martina Huber, and the University of Wageningen have used a new technique to monitor how certain proteins rearrange into a functional structure. With a smart tracing technique (DEER), the researchers studied the folding process of the protein flavodoxin. Inside a cell, each protein has to restructure itself by folding into a new arrangement. Errors in this process can cause several diseases, like cystic fibrosis and neurodegenerative diseases like Alzheimer’s. The results are presented in the Journal of Physical Chemistry.

Proteins play an important role in biological processes that also happen in our body. Each type of protein consists of a unique series of amino acids, which can take on numerous structures. To execute certain tasks—like electron transmission in photosynthesis, in the case of flavodoxin—a protein needs to fold into a specific structure. This is as essential for its function as the particles that it consists of. But where it is easy to determine the contents, it is far from clear how the ‘folding’ of proteins works. For this, we need information about the structure during multiple stages in the folding process. By understanding this process, scientists are able to see what happens when something goes wrong and a physical defect arises. In the end, this enables development of medicine against illnesses caused by errors in the folding process.

The Leiden research group used an existing technique for distinguishing materials in a sample: Electron Paramagnetic Resonance (EPR). Apart from radicals and certain metal ions, biophysicists can trace the atomic group nitroxide with EPR, and smart biochemical methods make it possible to attach the nitroxide group to a preferred site on the protein molecule. Lead author Martin van Son and his colleagues realized they could glue the nitroxide onto the protein to use it as a label to study the folding process. Just like an actor jumps around in front of a camera to make an animation movie, the protein folds in front of the EPR detector with labels attached to it. Since the distance between labels is monitored at each stage of the process, biophysicists can reconstruct a movie. During the development phase of the method, they only placed one set of labels on the protein, resulting in a low resolution. In the future, Huber will work with more labels, so she can see in higher resolution which part of the protein starts folding first.

Using EPR, you cannot record the whole movie in one go. The trick is to pause the folding process and take a snapshot. Unfortunately, it is not an option to simply resume the process from there. This is why the research team prepares multiple samples with proteins that haven’t completely folded yet, but instead have stopped at a different stage in the process. This stage depends on the concentration of denaturant that the researchers add to each sample. Now by measuring each sample, they take several snapshots that together form a movie. From the sequence of intermediate structures that a protein takes on before it finally arrives at its final shape, Huber deduces what reactions take place within the protein that induce folding.