Observing the structure of proteins in their natural environment is no easy task. It usually requires extracting, purifying and sometimes even crystallizing the proteins. A team from the CEA Frédéric-Joliot Life Science Institute has tested, for the first time, an Electron Paramagnetic Resonance (EPR) technique to determine the intramolecular distance in a protein inside intact bacterial cells of Escherichia coli. This groundbreaking technique will make it possible over time to solve the entire structure of this precise protein.

In this case, the EPR technique called PELDOR (Pulsed Electron Double Resonance) was used. "It detects the interaction between the electronic spins of two transition metal ions containing unpaired electrons, for instance between two gadolinium(III) ions or two manganese(II) ions," said Leandro Tabares, the last author of this study. "It thus becomes possible to measure the distance between two electronic spins and, consequently, between two metals." Distances between 2 and 15 nm can be accessed. "We have built, by genetic fusion, a protein which integrates two peptides on its surface, at both ends," Tabares said. "This peptide is quite special in that it can catch the gadolinium atoms that pass nearby." This genetically modified protein was designed to make sure it does not disturb the functioning of the living cell. It is, therefore, possible to measure the distance between two points of the protein with high accuracy, in its native cellular environment, without the need for purification or in vitro labelling. By varying the attachment points of the peptides on the protein, its structure can be approached. "It is the first time that we are capable of looking at a protein with an in-cell synthesized spin probe," Tabares added. All it takes is to put the cellular medium in contact with gadolinium for this metal to enter the cell, spontaneously attach to peptides and provide information on the distance between the two peptides of the protein.

So what is the next step? "Although we have not identified any toxic effect of gadolinium inside cells, we intend to work with another metal, manganese, found naturally in cells," Tabares said. "Of course, we are also looking at answering true biological questions and at probing different types of cells."