Many drugs target proteins. Understanding the dynamics of a protein and how it binds to its partners is thus essential for developing effective new therapeutic molecules. However, the complex lives of many proteins, their plasticity, and their ever-changing structure that depends on external parameters (partners, temperature, etc.) makes their study difficult. In addition, only some of their conformations are functional, just as only one type of key can open a lock. A team from the IBS, in collaboration with the EPFL¹ and ENS² of Lyon, has developed a novel method to study the dynamics of these very restless biological molecules. "The idea is to put a protein in a 'deep sleep' and then watch it slowly wake up until it becomes functional", says Martin Blackledge, laboratory director at the IBS.

Progressively waking up a protein from an inert to functional state

This deep sleep is achieved by lowering the temperature to -168°C, at which point the different protein components immobilize. By progressively increasing the temperature to 7°C, the researchers observed these components wake up one after the other under thermal agitation, similar to somebody in the early morning who first opens their eyes, then stretches, and finally gathers enough energy to get up. This experimental trick enables detecting the individual movements of the different components of a protein as well as the collective movements with a specially adapted NMR³ spectroscopy device. This was tested on GB1, a class of proteins that interact with antibodies. To mimic the protein environment in the cytoplasm of the cell, the researchers analyzed the protein surrounded by molecules of water. The latter were the first to move during the rise in temperature, at -113°C. The side chains of the protein then emerged from their lethargy, followed by its backbone at -53°C, at which temperature the protein becomes active. At each transition and throughout the warming stage, the NMR data helped to visualize the interaction between all parts of the protein. "This is the first time that a film has been reconstructed of a protein 'waking up', from an inert state at very low temperature to its functional state, including all intermediate steps", says the researcher. "We identified what temperature, and therefore what energy, is necessary to cross the barrier leading from one state to another."

NMR at all temperatures

NMR, which uses the magnetic properties of hydrogen, nitrogen and carbon atoms, is particularly difficult to apply to different protein configurations, which can be solid at low temperatures and liquid at room temperature. The IBS team thus developed a device devoted to variable temperatures that is capable of capturing an NMR signal over several days. For solid phases, the test tube containing the proteins must be inclined at a "magic" angle relative to the magnetic field, and then placed at rapid and constant rotation. In order to adapt to the rise in temperature and therefore the transition from solid to liquid phase, the system was equipped with a rotor that automatically and precisely adjusts the sample inclination angle.

This spectroscopy device has shown its power in reconstructing the film of a hydrated protein waking up. This experimental trick helps to finely analyze the movements of the different components of a protein, in order to understand how they fit together to make the protein functional.

Nuclear magnetic resonance (NMR) measured over a broad temperature range can analyze, depending on the amplitude and frequency of the movements, the dynamics of the different components of a protein. This helps understand how they fit together in order to make the protein functional.
© M. Blackledge

1. École Polytechnique Fédérale of Lausanne
2. École Normale Supérieure
3. Nuclear magnetic resonance