Neutron scattering and MD simulations together reveal the dynamics of the E. coli proteome at cell death temperature

What exactly happens inside a cell during thermal death was, until recently, a hotly debated question. Collaborative research led by Alessandro Paciaroni, Associate Professor at the University of Perugia, Judith Peters, Professor of Physics at the Université Grenoble Alpes and Fabio Sterpone, CNRS Research Director at the Theoretical Biochemistry Laboratory in Paris, combined state-of-the-art neutron scattering at the Institut Laue Langevin (ILL) with multiscale Molecular Dynamics (MD) simulations to reveal that only a minor fraction of the E. coli proteome unfolds at cell death provoking a striking dynamic slowdown and demonstrating the strong association between protein dynamics and bacterial metabolism and death.

Cells are the building blocks of life and proteins are responsible for nearly every cellular activity. Several thousand different proteins are contained within the cell and the activity of each is determined by how the protein is folded into a specific 3D structure. Though it was therefore understood that proteins play a key role during thermal cell death, theoretical models were contradictory, describing the unfolding of either a subset or the entire set – the proteome catastrophe model – of proteins within the cell.

Though considerable information exists about the structure of proteins, knowledge concerning their dynamic behaviour is relatively limited. The range of different motions with diverse time scales and amplitudes makes the study of protein dynamics highly complex. “Neutrons are a particularly valuable probe for the study of protein dynamics due to their ability to investigate unlabelled in vivo samples,” explains Paciaroni. “Other techniques, such as fluorescence spectroscopy, track dynamics by tagging the protein with an additional fluorescent protein. However, this alters the target protein, in this case by increasing the molecular weight, which could influence the dynamics.”

Previous research carried out by Paciaroni and Sterpone, published in the Proceedings of the National Academy of Sciences in 2017, combined neutron scattering acquired using the backscattering spectrometer IN13 at the ILL with MD simulations to study the relationship between protein dynamics and thermal stability. To extend their findings to bacteria (single-celled organisms), the researchers were joined by Peters who contributed extensive experience in elastic and quasi-elastic neutron scattering due to 14 years co-responsibility for IN13.

State-of-the-art Quasi-Elastic Neutron Scattering (QENS) experiments were carried out using the backscattering spectrometer IN16B instrument at the ILL by Daniele Di Bari, PhD student awarded funding by the French-Italian University (Université Franco Italienne/Università Italo Francese).

“QENS is the only technique that provides simultaneous access to both the time and length scales of the investigated system’s dynamics,” explains Peters. “And IN16B is the best high-resolution spectrometer in the world, the sub-micro eV energy resolution provides the nanosecond time scale necessary to study protein diffusive dynamics inside the cell.”