​There are many drugs which target proteins. Understanding the dynamics of a protein and how it links with its partners is therefore key to developing new and effective drug treatments. Many proteins have very complex lives, particularly in terms of their plasticity, since their structure is constantly changing and is dependent on external parameters (partner molecules, temperature, etc.), making them extremely difficult to study. Furthermore, this dynamic motion is essential for the protein to interact with its partner molecules and therefore, for it to be functional. A team at IBS (CEA/CNRS/Université Joseph Fourier), in collaboration with EPFL and ENS-Lyon, has developed an unprecedented method for studying the dynamics of these biological molecules and their extremely agitated motion. "The idea is to put a protein to sleep and then watch it gradually wake up until it becomes functionally active," explained Martin Blackledge, who heads the Dynamics and Flexibility by NMR Group at IBS.

To achieve such a deep sleep, the researchers froze the protein at -168°C, the temperature at which the different component parts of the protein become immobile. By gradually raising the temperature back up to 7°C, the team observed the protein's individual components waking up one after the other under the effects of thermal agitation, just like a person waking up who first opens his eyes, stretches and eventually mobilizes enough energy to get up. Thanks to this experimental technique, the researchers have been able to detect the individual motions of the different components that make up a protein, as well as their collective motions, using an NMR spectroscopy device specially-adapted and developed by the team at IBS. The experimental process was tested using GB1, a class of proteins that interact with antibodies. To mimic the protein's environment in the cell cytoplasm, the researchers analyzed the protein surrounded by water molecules. As the temperature rises, the water molecules are the first to become active, at
-113°C. The protein's side-chains are then shaken out of their sleepy state, followed by its backbone, at -53°C, the temperature at which the protein becomes active. As the temperature increases and at each stage of transition, the interactions between the different parts of the protein were observed thanks to the NMR data. "This is the first time we have been able to 'film' a protein 'waking up' with such precision, from an inert state at very low temperature to its functionally active state, reconstituting each stage along the way," said the researcher. "We have identified the temperature, and therefore the energy, at which the barrier from from one state to the other is crossed."

The NMR spectroscopy device used has thus demonstrated that it is powerful enough to reconstitute the sequence of a protein in a water solution 'waking up', a process that enables detailed analysis of the motions of the different protein components, enabling us to understand how they interact to make the protein functionally active.