The influence of pressure on the structure and protein-protein interaction potential of dense protein solutions was studied and analyzed using small-angle x-ray scattering in combination with a liquid state theoretical approach. The structural as well as the interaction parameters of dense lysozyme solutions are affected by pressure in a nonlinear way. The structural properties of water lead to a modification of the protein-protein interactions below 4 kbar, which might have significant consequences for the stability of proteins in extreme natural environments.

aWe study the kinetics of the liquid-liquid phase separation (LLPS) and its arrest in protein solutions exhibiting a lower critical solution temperature (LCST) phase behavior using the combination of ultrasmall angle X-ray scattering (USAXS) and very-small angle neutron scattering (VSANS). We employ a previously established model system consisting of bovine serum albumin (BSA) solutions with YCl 3 . We follow the phase transition from sub-second to 10 4 s upon an off-critical temperature jump. After a temperature jump, the USAXS profiles exhibit a peak that grows in intensity and shifts to lower q values

We present results from small-angle x-ray scattering data on the effect of high pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase separation region, and characterize the underlying intermolecular protein-protein interactions as a function of temperature and pressure in this region of phase space. A reentrant liquid-liquid phase separation region has been discovered at elevated pressures, which originates in the pressure dependence of the solvent-mediated protein-protein interactions.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.

In the presence of trivalent cations, negatively charged globular proteins show a rich phase behaviour including reentrant condensation, crystallisation, clustering and lower critical solution temperature metastable liquid-liquid phase separation (LCST-LLPS). Here, we present a systematic study on how different multivalent cations can be employed to tune the interactions and the associated phase behaviour of proteins. We focus our investigations on the protein bovine serum albumin (BSA) in the presence of HoCl 3 , LaCl 3 and YCl 3 . Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho 3+ is very strong, while the one induced by La 3+ is comparatively weak when comparing the data to BSA-Y 3+ systems based on our previous work. Using zeta potential and isothermal titration calorimetry (ITC) measurements, we establish different binding affinities of cations to BSA with Ho 3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the proteinprotein interaction induced by these cations. Our findings imply that subtle differences in cation properties can be a sensitive tool to fine-tune protein-protein interactions and phase behaviour in solution.

The Materials Imaging and Dynamics (MID) instrument at the European X-ray Free-Electron Laser (EuXFEL) facility is described. EuXFEL is the first hard X-ray free-electron laser operating in the MHz repetition range which provides novel science opportunities. The aim of MID is to enable studies of nano-structured materials, liquids, and soft- and hard-condensed matter using the bright X-ray beams generated by EuXFEL. Particular emphasis is on studies of structure and dynamics in materials by coherent scattering and imaging using hard X-rays. Commission of MID started at the end of 2018 and first experiments were performed in 2019.

This study explores the possibility of measuring the dynamics of proteins in solution using X-ray photon correlation spectroscopy (XPCS) at nearly diffraction-limited storage rings (DLSRs). We calculate the signal-to-noise ratio (SNR) of XPCS experiments from a concentrated lysozyme solution at the length scale of the hydrodynamic radius of the protein molecule. We take into account limitations given by the critical X-ray dose and find expressions for the SNR as a function of beam size, sample-to-detector distance and photon energy. Specifically, we show that the combined increase in coherent flux and coherence lengths at the DLSR PETRA IV yields an increase in SNR of more than one order of magnitude. The resulting SNR values indicate that XPCS experiments of biological macromolecules on nanometre length scales will become feasible with the advent of a new generation of synchrotron sources. Our findings provide valuable input for the design and construction of future XPCS beamlines at DLSRs.