Investigating protein solution dynamics using XPCS at an XFEL

by Frank Schreiber, frank.schreiber@uni-tuebingen.de, Institute of Applied Physics of University of Tübingen, Germany, on behalf of the entire collaboration [*]

Novel experiment opens up possibility of studying collective dynamics providing insight for better drug design.

Abstract

Proteins carry out a wide range of important roles in our bodies. Knowledge about how they move around and through the crowded environments of our cells and tissues, is key to understanding how they carry out these crucial functions. Experiments exploring protein dynamics however, often use samples that do not reflect the crowded and complex environments of the cell. At the MID instrument at European XFEL, scientists carried out X-ray photon correlation spectroscopy experiments to measure nanoscopic structural dynamics of protein solution samples. The combination of this technique with the high repetition rate of X-ray pulses generated by European XFEL opens up the unique possibility of studying collective dynamics in protein solutions with high concentration; particularly interesting in the context of intracellular transport in eukaryotic cells, and drug design.

Catalysts, transporters, gate-keepers, messengers – proteins carry out a vast range of different roles in our bodies. In addition to their structure, how they move through, and interact and react with their crowded and complex environment impacts their function. And yet, our understanding of the dynamics of processes such as protein diffusion and transport in cells and tissues is still lacking.

Protein dynamics in crowded media, especially diffusion and transport, are essential for biological processes and cellular function [1]. A comprehensive knowledge of how proteins behave in these environments is especially useful for example for the design of new drugs. When drugs enter our blood streams, it is the proteins that bind them and transport and distribute them around the body XFEL. How fast this happens, for example, is crucial information for understanding how long it takes for a drug to be dispersed, but also, in the case of for example cancer drugs, how and when toxicity levels begin to become too much as the amount of protein bound drug increases in the bloodstream. A comprehensive understanding of dynamics of these kinds of processes could inform more effective treatments with fewer side effects for example.

Many factors influence protein diffusion and transport within the crowded environment of our cells including crowding by other molecules, local hydration and water effects and temporary cluster formation with other proteins, all slowing down and hindering protein movement [2]. Studies also show that the dynamics of proteins in crowded cellular spaces, exhibit different behavior patterns to other systems. All of these need to be taken into account when modeling these systems. In highly concentrated environments, the dynamics significantly differ from that of a dilute system.

Experimental work studying protein dynamics has until now relied on a range of techniques, from energy-resolved neutron scattering to optical techniques and rheology, each with their own time and length scale range [1].

So far information on the collective dynamics at length scales comparable to the dimension of the proteins, and at time scales in the order of microseconds, where long-time diffusion acts, was hard to obtain. X-ray photon correlation spectroscopy combined with the fast repetition rates of the European XFEL can access these time scales and, importantly, processes such as cluster formation [3-5].

At the MID instrument at European XFEL, low dose X-ray photon correlation spectroscopy on antibody protein solutions containing immunoglobulin proteins was employed. The experiment delivered a wealth of information and new insights including how the diffusion dynamics changed in relation to the dose rate, and detailed insights into cluster formation changed over time and space [*].

This first protein MHz-XPCS experiment opens the door for investigating the microsecond time scale fast dynamics on microscopic length scales which is previously impossible to access by other techniques. Protein dynamics in this time and length scales are closely related to protein-protein interactions that determine reaction speeds, protein function, aggregation, phase separation and the solution viscosity with obvious implications for modern drug production and the design of drug delivery systems.

References

[*] M. Reiser et al., Resolving molecular diffusion and aggregation of antibody proteins with megahertz X-ray free-electron laser pulses, Nat. Commun. 13 (2022) 5528

[1]  M. Grimaldo et al., Dynamics of proteins in solution, Quart. Rev. Biophys. 52 (2019) e7, 1

[2]  F. Roosen-Runge et al., Protein self-diffusion in crowded solutions, PNAS 108 (2011) 11815

[3]  F. Perakis and C. Gutt, Towards Molecular Movies with X-Ray Photon Correlation Spectroscopy, Phys. Chem. Chem. Phys. 22, 19443 (2020).

[4]  F. Lehmkühleret al., Emergence of Anomalous Dynamics in Soft Matter Probed at the European XFEL, Proc. Natl. Acad. Sci. (2020).

[5]  F. Dallari et al., Analysis Strategies for MHz XPCS at the European XFEL, Appl. Sci. 11, 17 (2021).