Unfolding of protein ‘filmed’

'Snapshot' of the unfolding of the CylR2 protein from Enterococcus faecalis.

Alzheimer’s, Parkinson’s and Huntington’s chorea are all diseases caused by misfolded proteins that form insoluble clumps in the brains of affected persons and, finally, destroy their nerve cells. Proteins can perform their tasks properly only if their amino acid chains are folded correctly. But what exactly happens when proteins fold or unfold was previously nearly impossible to investigate. Conventional biological methods are not suitable for directly observing the unfolding process because with heat and pressure, proteins easily lose their shape and the intermediate forms that occur in the course of protein folding are much too transient.

Scientists at the Max Planck Institute for Biophysical Chemistry (MPIbpc) and the German Centre for Neurodegenerative Diseases (DZNE) in Göttingen in collaboration with scientists at the Polish Academy of Sciences in Warsaw and at the University of Warsaw have “filmed” for the first time, at atomic resolution, how a protein progressively “loses its shape”. The researchers reasoned that if a protein is slowly cooled down, its intermediate forms will accumulate in larger quantities than in commonly used denaturation methods. “We hoped that these quantities would be sufficient to examine the intermediate forms with nuclear magnetic resonance [NMR] spectroscopy,” said Markus Zweckstetter, a structural biologist and head of the research team.

His team chose a key protein (CylR2) for toxin production in Enterococcus faecalis, a pathogen frequently encountered in hospitals, as the research subject. Its three-dimensional shape made CylR2 a particularly promising candidate for investigation. “ClyR2 is a relatively small protein composed of two identical subunits. This gave us a great chance to be able to visualise the individual stages of its unfolding process in the test tube,” explained the chemists Mariusz and Lukasz Jaremko. They cooled the protein successively from 25° Celsius to -16° C and examined its intermediate forms with NMR spectroscopy. “We clearly see how the CylR2 protein ultimately splits into its two subunits. The individual subunit is initially relatively stable. With further cooling, the protein continues to unfold, and at -16 °C it is extremely unstable and dynamic. This unstable protein form provides the seed for folding and can also ‘trigger’ the misfolding,” says Zweckstetter. The findings may provide insights into how proteins assume their spatial structure and why intermediate forms misfold in the event of illness.