RESUMEN
Studying protein interactions at low temperatures has important implications for optimizing cryostorage processes of biological tissue, food, and protein-based drugs. One of the major issues is related to the formation of ice nanocrystals, which can occur even in the presence of cryoprotectants and can lead to protein denaturation. The presence of ice nanocrystals in protein solutions poses several challenges since, contrary to microscopic ice crystals, they can be difficult to resolve and can complicate the interpretation of experimental data. Here, using a combination of small- and wide-angle X-ray scattering (SAXS and WAXS), we investigate the structural evolution of concentrated lysozyme solutions in a cryoprotected glycerol-water mixture from room temperature (T = 300 K) down to cryogenic temperatures (T = 195 K). Upon cooling, we observe a transition near the melting temperature of the solution (T ≈ 245 K), which manifests both in the temperature dependence of the scattering intensity peak position reflecting protein-protein length scales (SAXS) and the interatomic distances within the solvent (WAXS). Upon thermal cycling, a hysteresis is observed in the scattering intensity, which is attributed to the formation of nanocrystallites in the order of 10 nm. The experimental data are well described by the two-Yukawa model, which indicates temperature-dependent changes in the short-range attraction of the protein-protein interaction potential. Our results demonstrate that the nanocrystal growth yields effectively stronger protein-protein attraction and influences the protein pair distribution function beyond the first coordination shell.
Asunto(s)
Hielo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Congelación , Solventes , Soluciones/químicaRESUMEN
Hydrated proteins undergo a transition in the deeply supercooled regime, which is attributed to rapid changes in hydration water and protein structural dynamics. Here, we investigate the nanoscale stress-relaxation in hydrated lysozyme proteins stimulated and probed by X-ray Photon Correlation Spectroscopy (XPCS). This approach allows us to access the nanoscale dynamics in the deeply supercooled regime (T = 180 K), which is typically not accessible through equilibrium methods. The observed stimulated dynamic response is attributed to collective stress-relaxation as the system transitions from a jammed granular state to an elastically driven regime. The relaxation time constants exhibit Arrhenius temperature dependence upon cooling with a minimum in the Kohlrausch-Williams-Watts exponent at T = 227 K. The observed minimum is attributed to an increase in dynamical heterogeneity, which coincides with enhanced fluctuations observed in the two-time correlation functions and a maximum in the dynamic susceptibility quantified by the normalized variance χT. The amplification of fluctuations is consistent with previous studies of hydrated proteins, which indicate the key role of density and enthalpy fluctuations in hydration water. Our study provides new insights into X-ray stimulated stress-relaxation and the underlying mechanisms behind spatiotemporal fluctuations in biological granular materials.
Asunto(s)
Proteínas , Agua , Rayos X , Proteínas/química , Temperatura , Agua/química , TermodinámicaRESUMEN
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.
Asunto(s)
Electrones , Rayos Láser , Inmunoglobulinas , Proteínas/química , Radiografía , Rayos XRESUMEN
The breakdown of Stokes-Einstein relation in liquid water is one of the many anomalies that take place upon cooling and indicates the decoupling of diffusion and viscosity. It is hypothesized that these anomalies manifest due to the appearance of nanometer-scale spatial fluctuations, which become increasingly pronounced in the supercooled regime. Here, we explore the validity of the Stokes-Einstein relation in supercooled water using nanomolecular probes. We capture the diffusive dynamics of the probes using dynamic light scattering and target dynamics at different length scales by varying the probe size, from ≈100 nm silica spheres to molecular-sized polyhydroxylated fullerenes (≈1 nm). We find that all the studied probes, independent of size, display similar diffusive dynamics with an Arrhenius activation energy of ≈23 kJ mol-1. Analysis of the diffusion coefficient further indicates that the probes, independent of their size, experience similar dynamic environment, which coincides with the macroscopic viscosity, while single water molecules effectively experience a comparatively lower viscosity. Finally, we conclude that our results indicate that the Stokes-Einstein relation is preserved for diffusion of probes in supercooled water T ≥ 260 K with size as small as ≈1 nm.
RESUMEN
Understanding the mechanism responsible for the protein low-temperature crossover observed at T≈ 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 Å-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.