Realization of low dose XPCS experiments for the investigation of protein dynamics

In the cell cytoplasm proteins are surrounded by water and a crowd of macromolecules. For a quantitative understanding of the cellular system knowledge of the dynamic processes in this crowded environment is of key importance. Reversible formation of protein condensates guarantee vitally essential functions. Irreversible protein aggregation processes are responsible for protein condensation diseases such as sickle cell anemia. Furthermore protein dynamics in concentrated solutions are relevant for the design of future drugs and important in the food industry. Up to now, static properties of proteins in crowded environments are well investigated, but little is known about their dynamics.
X-ray photon correlation spectroscopy (XPCS) provides access to both the spatial and temporal properties of the sample simultaneously. Using new X-ray sources such as forth generation storage rings, XPCS experiments on time scales from femtoseconds to hours will be feasible. However, the highly intense X-ray beam is also the cause of radiation damage which requires to develop and implement new avenues of low dose XPCS experiments.
This thesis demonstrates the feasibility of Bio-XPCS measurements to investigate protein dynamics during a liquid-liquid phase separation on a hierarchy of length scales from droplet sizes in the region of micrometers down to the single protein length scale at nanometers. Established strategies to mitigate radiation damage, such as cyro-cooling, are not feasible for investigating protein dynamics. Therefore we increased the beam size leading to a reduced photon density on the sample. To optimize the signal-to-noise ratio we employed a large sample-to-detector distance in the USAXS setup at the P10 Coherence Application Beamline at PETRA III to resolve the speckles.
The experiments, performed at the Deutsches Elektronen-Synchrotron (DESY), revealed the dynamics of a protein model system composed of the antibody IgG in presence of polyethylene glycol (PEG) during the liquid-liquid phase separation (LLPS). On the protein droplet scale dynamics caused by the interface formation of the LLPS followed by heterogeneous ballistic motion of the protein domains due to interfacial coarsening were identified. Furthermore highly heterogeneous dynamics associated with particle clusters inside the protein rich domains were found on the length scale of nanometers.
Further research regarding protein condensation diseases and the design of future protein drugs will benefit from the methods developed in this thesis by making Bio-XPCS experiments at the new X-ray sources feasible.