Michelle Dargasz

Macromolecular crowding plays a crucial role in modulating protein dynamics in cellular and in vitro environments. Polymeric crowders such as dextran and Ficoll are known to induce entropic forces, including depletion interactions, that promote structural organization, yet their nanoscale consequences for protein dynamics remain poorly understood. Here, we employ megahertz X-ray photon correlation spectroscopy (MHz-XPCS) at the European X-ray Free Electron Laser (XFEL) to probe the dynamics of the protein ferritin in solutions containing sucrose, dextran, and Ficoll. We find pronounced changes in collective protein dynamics in polymeric crowders, revealing depletion-driven short-range attractions that, combined with long-range repulsions, give rise to intermediate-range organization. These mesoscale correlations undergo collective relaxation on microsecond to millisecond timescales, as directly resolved by XPCS through the decay of ferritin density fluctuations. The magnitude of this depends sensitively on crowder molecular weight and type. Normalizing the crowder concentration by c * reveals scaling behavior of ferritin self-diffusion with a crossover near 2 c *, marking a transition from depletion-enhanced mobility to viscosity-dominated slowing. Our results demonstrate that bulk properties alone are insufficient to describe protein dynamics in crowded solutions, highlighting the need to include polymer-specific interactions and depletion theory in models of crowded environments.

In this article, we present the experimental protocol and data-processing framework for megahertz X-ray Photon Correlation Spectroscopy (MHz-XPCS) experiments on soft matter samples implemented at the Materials Imaging and Dynamics (MID) instrument of the European X-ray Free-Electron Laser (EuXFEL). Due to the introduction of a standard configuration and the implementation of a highly automated data-processing pipeline, MHz-XPCS measurements can now be conducted and analyzed with minimal user intervention. A key challenge lies in managing the extremely large data volumes generated by the Adaptive Gain Integrating Pixel Detector (AGIPD) – often reaching several petabytes within a single experiment. We describe the technical implementation, discuss the hardware requirements related to effective parallel data processing and propose strategies to enhance data quality, in particular related to data reduction strategies and an improvement of the signal-to-noise ratio. Finally, we address strategies for making the processed data FAIR (Findable, Accessible, Interoperable, Reusable), in alignment with the goals of the DAPHNE4NFDI project.

The effect of lowered pH on the thermal gelation behavior of hen egg yolk was investigated over the temperature range of 58–72 °C using low-dose X-ray photon correlation spectroscopy in ultra-small-angle X-ray scattering geometry. Progressive structural and dynamical alterations were observed at room temperature with decreasing pH, indicative of acid-induced protein denaturation, which correlates with an increase in yolk viscosity. Temperature- and pH-dependent structural and dynamic investigation suggests an acceleration in gel formation with decreasing pH. The time–temperature superposition relationship observed in all samples suggests an identical mechanism underlying protein aggregation–gelation with a temperature-dependent reaction rate. The sol–gel transition time extracted from kinetic and dynamic information follows Arrhenius behavior, with no significant change in the activation energy (450 ± 20 kJ/mol) of gelation. However, the prefactor A systematically decreases with decreasing pH, indicating that acidification accelerates gelation primarily by increasing the frequency of productive encounters without altering the fundamental energy barrier of the process.

Low-density lipoproteins (LDLs) are central to nutrient transport in egg yolk and have emerged as natural nanocarriers for drug delivery. Their biological function critically depends on mobility within densely crowded environments, yet the mechanisms governing their motion remain elusive, largely because conventional techniques cannot access the relevant microsecond timescales. Here, we employ megahertz X-ray photon correlation spectroscopy at the European X-ray Free Electron Laser facility to resolve LDL dynamics in native yolk-plasma. This approach reveals transient caging and memory effects and shows that the combined influence of particle softness and hydrodynamic coupling slows diffusion by nearly two orders of magnitude compared to dilute solutions. However, this reduction could not be scaled with an increase in macroscopic viscosity obtained from rheometry, indicating deviations from the Stokes–Einstein relation. Despite this slowdown, yolk-plasma remains a “sluggish yet liquid state”, balancing dense packing and the fluidity required for lipid release during embryonic development. These results establish a quantitative framework connecting microstructure, hydrodynamics, and transport in crowded soft-matter systems, with implications for developmental biology and nanomedicine.

Understanding protein motion within the cell is crucial for predicting reaction rates and macromolecular transport in the cytoplasm. A key question is how crowded environments affect protein dynamics through hydrodynamic and direct interactions at molecular length scales. Using megahertz X-ray Photon Correlation Spectroscopy (MHz-XPCS) at the European X-ray Free Electron Laser (EuXFEL), we investigate ferritin diffusion at microsecond time scales. Our results reveal anomalous diffusion, indicated by the non-exponential decay of the intensity autocorrelation function g ₂ ( q , t ) at high concentrations. This behavior is consistent with the presence of cage-trapping between the short- and long-time protein diffusion regimes. Modeling with the δ γ -theory of hydrodynamically interacting colloidal spheres successfully reproduces the experimental data by including a scaling factor linked to the protein direct interactions. These findings offer insights into the complex molecular motion in crowded protein solutions, with potential applications for optimizing ferritin-based drug delivery, where protein diffusion is the rate-limiting step.

This study focuses on the kinetics of light-induced mesophase transitions in lyotropic liquid crystals containing a mixture of phospholipids and azobenzene amphiphiles. Lipid membranes organize in a wide range of morphologies, directly influencing their functionality and the efficiency of associated components such as proteins. Transitions between mesophases occur naturally during membrane fusion and can also be triggered by multiple factors, such as pH, salinity, temperature and light. Employing light to isomerize artificial photoswitchable lipids in mixed model membranes containing 1,2-dipalmitoylphosphatidylcholine or 1,2-didecanoylphosphatidylcholine revealed light-induced structural changes including mesophase transitions from a lamellar to a cubic Pn3m phase. Performing time-resolved small-angle X-ray scattering measurements, the kinetics of the change in membrane repeat distance and the transition from a lamellar to a bicontinuous cubic phase could be captured on the timescale of tens of seconds. The results demonstrate new possibilities for investigating intermediate states during mesophase transitions that are important to understand membrane fusion, and they highlight the potential of photoswitchable lipids for designing bespoke drug delivery systems.

We investigated the effect of the NaCl concentration (0.3–2M) on the structure and dynamics of hen egg yolk at room temperature and during thermal gelation at temperatures in the range of 66–90 °C utilizing low-dose x-ray photon correlation spectroscopy in ultra-small angle x-ray scattering geometry. With an increase in the salt concentration, we observe progressive structural and dynamic changes at room temperature, indicating the disruption of yolk components such as yolk-granules and yolk-plasma proteins. Temperature- and salt-dependent structural and dynamic investigations suggest a delay in the gel formation and aggregation of yolk low-density lipoproteins with increasing ionic strength. However, the time–temperature superposition relationship observed in all samples suggests an identical mechanism underlying protein aggregation–gelation with a temperature-dependent reaction rate. The sol–gel transition time extracted from kinetic and dynamic information follows Arrhenius’s behavior, and the activation energy (460 kJ/mol) is found to be independent of the salt concentration.

The functionality and efficiency of proteins within a biological membrane are highly dependent on both the membrane lipid composition and the physiochemical properties of the solution. Lipid mesophases are directly influenced by changes in temperature, pH, water content or due to individual properties of single lipids such as photoswitchability. In this work, we were able to induce light- and temperature-driven mesophase transitions in a model membrane system containing a mixture of 1,2-dipalmitoyl-phosphatidylcholine phospholipids and azobenzene amphiphiles. We observed reversible and reproducible transitions between the lamellar and Pn3m cubic phase after illuminating the sample for 5 min with light of 365 and 455 nm wavelengths, respectively, to switch between the cis and trans states of the azobenzene N=N double bond. These light-controlled mesophase transitions were found for mixed complexes with up to 20% content of the photosensitive molecule and at temperatures below the gel-to-liquid crystalline phase transition temperature of 33°C. Our results demonstrate the potential to design bespoke model systems to study the response of membrane lipids and proteins upon changes in mesophase without altering the environment and thus provide a possible basis for drug delivery systems.

The soft-grainy microstructure of cooked egg yolk is the result of a series of out-of-equilibrium processes of its protein-lipid contents; however, it is unclear how egg yolk constituents contribute to these processes to create the desired microstructure. By employing X-ray photon correlation spectroscopy, we investigate the functional contribution of egg yolk constituents: proteins, low-density lipoproteins (LDLs), and yolk-granules to the development of grainy-gel microstructure and microscopic dynamics during cooking. We find that the viscosity of the heated egg yolk is solely determined by the degree of protein gelation, whereas the grainy-gel microstructure is controlled by the extent of LDL aggregation. Overall, protein denaturation-aggregation-gelation and LDL-aggregation follows Arrhenius-type time-temperature superposition (TTS), indicating an identical mechanism with a temperature-dependent reaction rate. However, above 75 °C TTS breaks down and temperature-independent gelation dynamics is observed, demonstrating that the temperature can no longer accelerate certain non-equilibrium processes above a threshold value.