While the interplay between liquid-liquid phase separation (LLPS) and glass formation in biological systems is highly relevant for their structure formation and thus function, the exact underlying mechanisms are not well known. The kinetic arrest originates from the slowdown at the molecular level, but how this propagates to the dynamics of microscopic phase domains is not clear. Since with diffusion, viscoelasticity, and hydrodynamics, distinctly different mechanisms are at play, the dynamics needs to be monitored on the relevant time and length scales and compared to theories of phase separation. Using x-ray photon correlation spectroscopy, we determine the LLPS dynamics of a model protein solution upon low temperature quenches and find distinctly different dynamical regimes. We observe that the early stage LLPS is driven by the curvature of the free energy and speeds up upon increasing quench depth. In contrast, the late stage dynamics slows down with increasing quench depth, fingerprinting a nearby glass transition. The dynamics observed shows a ballistic type of motion, implying that viscoelasticity plays an important role during LLPS. We explore possible explanations based on the Cahn-Hilliard theory with nontrivial mobility parameters and find that these can only partially explain our findings.
The kinetics of heat-induced gelation and the microscopic dynamics of a hen egg white gel are probed using x-ray photon correlation spectroscopy along with ultrasmall-angle x-ray scattering. The kinetics of structural growth reveals a reaction-limited aggregation process with a gel fractal dimension of ≈2 and an average network mesh size of ca. 400 nm. The dynamics probed at these length scales reveals an exponential growth of the characteristic relaxation times followed by an intriguing steady state in combination with a compressed exponential correlation function and a temporal heterogeneity. The degree of heterogeneity increases with decreasing length scale. We discuss our results in the broader context of experiments and models describing attractive colloidal gels.
The interplay of the glass transition with liquid− liquid phase separation (LLPS) is a subject of intense debate. We use the scattering invariant Q to probe how approaching the glass transition affects the shape of LLPS boundaries in the temperature/volume fraction plane. Two protein systems featuring kinetic arrest with a lower and an upper critical solution temperature phase behavior, respectively, are studied varying the quench depth. Using Q we noninvasively identify system-dependent differences for the effect of glass formation on the LLPS boundary. The glassy dense phase appears to enter the coexistence region for the albumin−YCl 3 system, whereas it follows the equilibrium binodal for the γ-globulin−PEG system.
Microscopic dynamics
of complex fluids in the early stage of spinodal
decomposition (SD) is strongly intertwined with the kinetics of structural
evolution, which makes a quantitative characterization challenging.
In this work, we use X-ray photon correlation spectroscopy to study
the dynamics and kinetics of a protein solution undergoing liquid–liquid
phase separation (LLPS). We demonstrate that in the early stage of
SD, the kinetics relaxation is up to 40 times slower than the dynamics
and thus can be decoupled. The microscopic dynamics can be well described
by hyper-diffusive ballistic motions with a relaxation time exponentially
growing with time in the early stage followed by a power-law increase
with fluctuations. These experimental results are further supported
by simulations based on the Cahn–Hilliard equation. The established
framework is applicable to other condensed matter and biological systems
undergoing phase transitions and may also inspire further theoretical
work.
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.
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