Understanding the molecular dynamics and diffusivity of proteins at cryogenic temperatures is essential for optimizing cryopreservation techniques of biological materials, with applications ranging from biotechnology to food science. This knowledge is also relevant for organism living under extreme conditions, such as the sub-zero temperatures of the Arctic Sea. A key feature observed at cryogenic temperatures is the protein dynamic transition near T = 230 K, where proteins lose their flexibility and functionality. The nature of this transition is still elusive, also due to the experimental challenge posed from the crystallization of water at these low temperatures. We investigate the structural dynamics of proteins under supercooled conditions with two approaches: hydrated proteins, where the absence of bulk water prevents freezing, and cryoprotected protein solutions, where cryoprotectants lower the water melting point.

We employ existing X-ray scattering techniques, namely small- and wide- angle X-ray scattering. Additionally, we advance the development of X-ray Photon Correlation Spectroscopy for studying biological systems. In hydrated lysozyme, we observe water temperature-dependent structural changes with a crossover at T = 230 K. Notably, nanoscale dynamics of hydrated proteins reveal enhanced density fluctuations at the same temperature, consistent with the crossing of the hypothesized Widom line in bulk water. This finding suggest a clear link between the protein dynamic transition and the water properties. We extend these studies to cryoprotected ferritin solutions. We explore the collective dynamics of proteins at molecular length scales and observed anomalous diffusion, which was enhanced with increasing protein concentration. Furthermore, we detect a deviation from the Stokes-Einstein relation and a shift in the arrest temperature of the solvent to lower temperature, likely caused by the presence of proteins, which significantly alter the local solvent environment. These results suggests that protein mobility near glassy conditions and at supercooled temperatures may differ drastically from predictions based on solvent viscosity.  

Recent experiments and theoretical studies strongly indicate that water exhibits a liquid-liquid phase transition (LLPT) in the supercooled domain. An open question is how the LLPT of water can affect the properties of aqueous solutions. Here, we study the structural and thermodynamic properties of supercooled glycerol-water microdroplets at dilute conditions (χg = 3.2% glycerol mole fraction). The combination of rapid evaporative cooling with femtosecond X-ray scattering allows us to outrun crystallization and gain access to the deeply supercooled regime down to T = 229.3 K. We find that the density fluctuations of the glycerol-water solution or, equivalently, its isothermal compressibility, κT, increases upon cooling. This is confirmed by molecular dynamics simulations, which indicate that the presence of glycerol shifts the temperature of maximum κT from T = 230 K in pure water down to T = 223 K in the solution. Our findings elucidate the interplay between the complex behavior of water, including its LLPT, and the properties of aqueous solutions at low temperatures, which can have practical consequences in cryogenic biological applications and cryopreservation techniques.

Hydrated proteins undergo a transition in the deeplysupercooledregime, which is attributed to rapid changes in hydration water andprotein structural dynamics. Here, we investigate the nanoscale stress-relaxationin hydrated lysozyme proteins stimulated and probed by X-ray PhotonCorrelation Spectroscopy (XPCS). This approach allows us to accessthe nanoscale dynamics in the deeply supercooled regime (T = 180 K), which is typically not accessible through equilibriummethods. The observed stimulated dynamic response is attributed tocollective stress-relaxation as the system transitions froma jammed granular state to an elastically driven regime. The relaxationtime constants exhibit Arrhenius temperature dependence upon coolingwith a minimum in the Kohlrausch-Williams-Watts exponentat T = 227 K. The observed minimum is attributedto an increase in dynamical heterogeneity, which coincides with enhancedfluctuations observed in the two-time correlation functions and amaximum in the dynamic susceptibility quantified by the normalizedvariance chi( T ). The amplification offluctuations is consistent with previous studies of hydrated proteins,which indicate the key role of density and enthalpy fluctuations inhydration water. Our study provides new insights into X-ray stimulatedstress-relaxation and the underlying mechanisms behind spatiotemporalfluctuations in biological granular materials.

Studying protein interactions at low temperatures hasimportantimplications for optimizing cryostorage processes of biological tissue,food, and protein-based drugs. One of the major issues is relatedto the formation of ice nanocrystals, which can occur even in thepresence of cryoprotectants and can lead to protein denaturation.The presence of ice nanocrystals in protein solutions poses severalchallenges since, contrary to microscopic ice crystals, they can bedifficult to resolve and can complicate the interpretation of experimentaldata. Here, using a combination of small- and wide-angle X-ray scattering(SAXS and WAXS), we investigate the structural evolution of concentratedlysozyme solutions in a cryoprotected glycerol-water mixturefrom room temperature (T = 300 K) down to cryogenictemperatures (T = 195 K). Upon cooling, we observea transition near the melting temperature of the solution (T & AP; 245 K), which manifests both in the temperaturedependence of the scattering intensity peak position reflecting protein-proteinlength scales (SAXS) and the interatomic distances within the solvent(WAXS). Upon thermal cycling, a hysteresis is observed in the scatteringintensity, which is attributed to the formation of nanocrystallitesin the order of 10 nm. The experimental data are well described bythe two-Yukawa model, which indicates temperature-dependent changesin the short-range attraction of the protein-protein interactionpotential. Our results demonstrate that the nanocrystal growth yieldseffectively stronger protein-protein attraction and influencesthe protein pair distribution function beyond the first coordinationshell.

Here, we demonstrate an experimental proof of concept for nanofocused x-ray photon correlation spectroscopy, a technique sensitive to nanoscale fluctuations present in a broad range of systems. The experiment, performed at the NanoMAX beamline at MAX IV, uses a novel event-based x-ray detector to capture nanoparticle structural dynamics with microsecond resolution. By varying the nanobeam size from σ=88 nm to σ=2.5μm, we quantify the effect of the nanofocus on the small-angle scattering lineshape and on the diffusion coefficients obtained from nano-XPCS. We observe that the use of nanobeams leads to a multifold increase in speckle contrast, which greatly improves the experimental signal-to-noise ratio, quantified from the two-time intensity correlation functions. We conclude that it is possible to account for influence of the high beam divergence on the lineshape and measured dynamics by including a convolution with the nanobeam profile in the model.

X-ray free-electron lasers (XFELs) with megahertz repetition rate can provide novel insights into structural dynamics of biological macromolecule solutions. However, very high dose rates can lead to beam-induced dynamics and structural changes due to radiation damage. Here, we probe the dynamics of dense antibody protein (Ig-PEG) solutions using megahertz X-ray photon correlation spectroscopy (MHz-XPCS) at the European XFEL. By varying the total dose and dose rate, we identify a regime for measuring the motion of proteins in their first coordination shell, quantify XFEL-induced effects such as driven motion, and map out the extent of agglomeration dynamics. The results indicate that for average dose rates below 1.06 kGy μs⁻¹ in a time window up to 10 μs, it is possible to capture the protein dynamics before the onset of beam induced aggregation. We refer to this approach as correlation before aggregation and demonstrate that MHz-XPCS bridges an important spatio-temporal gap in measurement techniques for biological samples.

Understanding the mechanism responsible for the protein low-temperature crossover observed at T approximate to 220 K can help us improve current cryopreservation technologies. This crossover is associated with changes in the dynamics of the system, such as in the mean-squared displacement, whereas experimental evidence of structural changes is sparse. Here we investigate hydrated lysozyme proteins by using a combination of wide-angle X-ray scattering and molecular dynamics (MD) simulations. Experimentally we suppress crystallization by accurate control of the protein hydration level, which allows access to temperatures down to T = 175 K. The experimental data indicate that the scattering intensity peak at Q = 1.54 angstrom(-1), attributed to interatomic distances, exhibits temperature-dependent changes upon cooling. In the MD simulations it is possible to decompose the water and protein contributions and we observe that, while the protein component is nearly temperature independent, the hydration water peak shifts in a fashion similar to that of bulk water. The observed trends are analysed by using the water-water and water-protein radial distribution functions, which indicate changes in the local probability density of hydration water.

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 g₂(q,t) at high concentrations. This behavior is consistent with the presence of cage-trapping in between the short- and long-time protein diffusion regimes. Modeling with the δγ-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 new 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.

It is proposed that a liquid-liquid transition (LLT), related to the hypothesized transition between high- and low-density liquids (HDL, LDL) in pure water, also exists in supercooled aqueous mixtures. However, experimental observations of the LLT in the supercooled solution is often complicated by the overlap with freezing. Here, we conducted an experiment probing the hypothesized LLT in deeply supercooled 16.5 mol% glycerol-water solution, combining X-ray photon correlation spectroscopy (XPCS), ultra small-angle X-ray scattering (USAXS) and wide-angle X-ray scattering (WAXS). This approach allows us to capture simultaneous, discontinuous structural and dynamic changes within the supercooled liquid following quenching to cryogenic temperatures (172-182 K). We observe changes in the inter-atomic liquid structure (from WAXS) as well as in the nanoscale structure and dynamics (from USAXS/XPCS), resembling a first-order LLT between HDL-like to LDL-like liquid. Importantly, we find that the LLT precedes the onset of ice crystalliization, which we can distinguish based on the advent of ice bragg peaks in WAXS. In addition, analysis of the two-time correlation (TTC) function from XPCS enables us to follow the dynamics during the LLT, which indicates super-diffusive ballistic-like motion and a gradual slowdown towards an arrested state upon freezing, consistent with an LLT via spinodal decomposition. We conclude that these results indicate the existence of a first-order LLT in supercooled glycerol-water solutions at intermediate glycerol concentrations, similar to that hypothesized for pure water at elevated pressures.

The liquid-liquid critical point (LLCP) hypothesis of water suggests that water exists in two structurally distinct liquid states, high- and low-density liquid (HDL, LDL), with an LLCP hidden in the supercooled regime at elevated pressures. However, its consequences for solvation and structural dynamics in aqueous solutions remain to be explored. Here, we probe the structure and thermodynamics of deeply supercooled microdroplets of prototypical aqueous solutions of glycerol. The combination of rapid evaporative cooling with ultrafast small- and wide-angle X-ray scatter-ing (SAXS, WAXS) allows us to outrun crystallization and gain access to the largely unexplored deeply supercooled dilute regime (3.2 mol% glycerol) down to T ≈ 229 K, which is not accessible by conventional cooling methods. The experimental results, and complementary molecular dynamics(MD) simulations, indicate an increase in the tetrahedral coordination and enhancement of HDL-and LDL-like density fluctuations upon supercooling. In addition, the extended temperature range of the MD simulations reveals a maximum in the isothermal compressibility at T ≈ 220 K, indicating the location of a Widom line shifted to slightly lower temperatures compared to that of pure water. We conclude that the apparent effect of the presence of glycerol molecules on the water hydrogen-bond structure resembles that of pressure. This opens the possibility to search for the existence of an LLCP in these aqueous solutions simply by varying the solute concentration.