Anna Stradner

Equilibrium cluster formation in concentrated protein solutions and colloids.

Controlling interparticle interactions, aggregation and cluster formation is of central importance in a number of areas, ranging from cluster formation in various disease processes to protein crystallography and the production of photonic crystals. Recent developments in the description of the interaction of colloidal particles with short-range attractive potentials have led to interesting findings including metastable liquid–liquid phase separation and the formation of dynamically arrested states (such as the existence of attractive and repulsive glasses, and transient gels). The emerging glass paradigm has been successfully applied to complex soft-matter systems, such as colloid–polymer systems and concentrated protein solutions. However, intriguing problems like the frequent occurrence of cluster phases remain. Here we report small-angle scattering and confocal microscopy investigations of two model systems: protein solutions and colloid–polymer mixtures. We demonstrate that in both systems, a combination of short-range attraction and long-range repulsion results in the formation of small equilibrium clusters. We discuss the relevance of this finding for nucleation processes during protein crystallization, protein or DNA self-assembly and the previously observed formation of cluster and gel phases in colloidal suspensions.

Internal structure and colloidal behaviour of covalent whey protein microgels obtained by heat treatment

Covalently cross-linked whey protein microgels (WPM) were produced without the use of a chemical cross-linking agent. The hierarchical structure of WPM is formed by a complex interplay of heat denaturation, aggregation, electrostatic repulsion, and formation of disulfide bonds. Therefore, well-defined spherical particles with a diameter of several hundreds of nanometers and with relatively low polydispersity are formed in a narrow pH regime (5.8–6.2) only. WPM production was carried out on large scale by heating a protein solution in a plate-plate heat exchanger. Thereafter, the microgels were concentrated by microfiltration and spray dried into a powder. The spherical structure of the WPM was conserved in the powder. After re-dispersion, the microgel dispersions fully recovered their initial structure and size distribution. Due to the formation of disulfide bonds the particles were internally covalently cross-linked and were remarkably stable in a large pH range. Because of the pH dependent charge of the constituents the particles underwent significant size changes upon shifting the pH. Small angle X-ray scattering experiments were used to reveal their internal structure, and we report on the pH-induced structural changes occurring on different length scale. Our experiments showed that close analogies could be drawn to internally cross-linked and pH-responsive microgels based on weak polyelectrolytes. WPM also exhibited a pronounced swelling at pH values below the isoelectric point (IEP), and a collapse at the IEP. However, in contrast to classical microgels, WPM are not build up by simple polymer chains but possess a complex hierarchical structure consisting of strands formed by clusters of aggregated denatured proteins that act as primary building blocks. They were flexible enough to respond to changes of the environment, and were stable enough to tolerate pH values where the proteins were highly charged and the strands were stretched.

Interplay between Spinodal Decomposition and Glass Formation in Proteins Exhibiting Short-Range Attractions

We investigate the competition between spinodal decomposition and dynamical arrest using aqueous solutions of the globular protein lysozyme as a model system for colloids with short-range attractions. We show that quenches below a temperature Ta lead to gel formation as a result of a local arrest of the protein-dense phase during spinodal decomposition. The rheological properties of these gels allow us to use centrifugation experiments to determine the local densities of both phases and to precisely locate the gel boundary and the attractive glass line close to and within the unstable region of the phase diagram.

Modeling equilibrium clusters in lysozyme solutions

We present a combined experimental and numerical study of the equilibrium cluster formation in globular-protein solutions under no-added salt conditions. We show that a cluster phase emerges as a result of a competition between a long-range screened Coulomb repulsion and a short-range attraction. A simple effective potential, in which electrostatic repulsion is fixed by experimental conditions and attraction is modeled with a generalized Lennard-Jones potential, accounts in a remarkable way for the wavevector dependence of the X-ray scattering structure factor.

A simple patchy colloid model for the phase behavior of lysozyme dispersions

We propose a minimal model for spherical proteins with aeolotopic pair interactions to describe the equilibrium phase behavior of lysozyme. The repulsive screened Coulomb interactions between the particles are taken into account assuming that the net charges are smeared out homogeneously over the spherical protein surfaces. We incorporate attractive surface patches, with the interactions between patches on different spheres modeled by an attractive Yukawa potential. The parameters entering the attractive Yukawa potential part are determined using information on the experimentally accessed gas-liquid-like critical point. The Helmholtz free energy of the fluid and solid phases is calculated using second-order thermodynamic perturbation theory. Our predictions for the solubility curve are in fair agreement with experimental data. In addition, we present new experimental data for the gas-liquid coexistence curves at various salt concentrations and compare these with our model calculations. In agreement with earlier findings, we observe that the strength and the range of the attractive potential part only weakly depend on the salt content.

Cluster-Driven Dynamical Arrest in Concentrated Lysozyme Solutions

We present a detailed experimental and numerical study of the structural and dynamical properties of salt-free lysozyme solutions. In particular, by combining small-angle X-ray scattering (SAXS) data with neutron spin echo (NSE) and rheology experiments, we are able to identify that an arrest transition takes place at intermediate densities, driven by the slowing down of the cluster motion. Using an effective pair potential among proteins, based on the combination of short-range attraction and long-range repulsion, we account remarkably well for the peculiar volume fraction dependence of the effective structure factor measured by SAXS. We show that a transition from a monomer to a cluster-dominated fluid happens at volume fractions larger than ϕ ≳ 0.05 where the close agreement between NSE measurements and Brownian dynamics simulations confirms the transient nature of the clusters. Clusters even stay transient above the geometric percolation found in simulation at ϕ > 0.15, though NSE reveals a cluster lifetime that becomes increasingly large and indicates a divergence of the diffusivity at ϕ ≃ 0.26. Macroscopic measurements of the viscosity confirm this transition where the long-lived-nature of the clusters is at the origin of the simultaneous dynamical arrest at all length scales.

New insight into cataract formation : Enhanced stability through mutual attraction

Small-angle neutron scattering experiments and molecular dynamics simulations combined with an application of concepts from soft matter physics to complex protein mixtures provide new insight into the stability of eye lens protein mixtures. Exploring this colloid-protein analogy we demonstrate that weak attractions between unlike proteins help to maintain lens transparency in an extremely sensitive and nonmonotonic manner. These results not only represent an important step towards a better understanding of protein condensation diseases such as cataract formation, but provide general guidelines for tuning the stability of colloid mixtures, a topic relevant for soft matter physics and industrial applications.

Density dependent interactions and structure of charged colloidal dispersions in the weak screening regime

We determine the structure of charge-stabilized colloidal suspensions at low ionic strength over an extended range of particle volume fractions using a combination of light and small angle neutron scattering experiments. The variation of the structure factor with concentration is analyzed within a one-component model of a colloidal suspension. We show that the observed structural behavior corresponds to a nonmonotonic density dependence of the colloid effective charge and the mean interparticle interaction energy. Our findings are corroborated by similar observations from primitive model computer simulations of salt-free colloidal suspensions.

The pH induced sol-gel transition in skim milk revisited. a detailed study using time-resolved light and X-ray scattering experiments

We present a detailed study of the evolution of the size, structure and stability of casein micelles upon acidification of skim milk typically applied in yogurt-making processes using a combination of time-resolved light and small-angle X-ray scattering experiments. While most of the available light scattering studies on casein acidification have been restricted to transparent and therefore highly diluted samples, we now profit from a newly developed multiangle 3D light scattering instrument, which allows for time-resolved measurements in highly turbid samples. Our experiments clearly demonstrate the presence of two parallel pH-dependent processes, micellar reassembly and aggregation. Using a systematic investigation of the effect of casein concentration, acidification rate, and ionic strength, we are able to decouple these two processes and obtain detailed information about the pH-induced restructuration of the casein micelle structure that occurs prior to destabilization. Moreover, our experiments also unambiguously demonstrate that these micellar reassembly processes are highly concentration dependent, and that typical light scattering studies conducted under highly diluted conditions are resulting in findings that may not be relevant for the situation encountered in industrial processes at higher concentrations. Experiments conducted with covalently cross-linked micelles, where the pH-induced reassembly has been suppressed, further confirm our findings.

Colloidal characterization and thermodynamic stability of binary eye lens protein mixtures.

We present a study of binary mixtures of eye lens crystallin proteins. A coarse-grained model of aqueous alpha- and gamma-crystallin mixtures based on molecular dynamics simulations and SANS experiments is proposed. Thermodynamic perturbation theory is implemented to obtain the stability boundaries, or spinodal surface, of the binary mixture in the full parameter space. The stability of these high-concentration crystallin mixtures was found to depend on the alpha-gamma attraction in a manner that is both extremely sensitive and nonmonotonic; stronger or weaker attraction resulted in a spectacularly enhanced instability. The relevance of these mechanisms as possible sources of the alteration of the spatial distribution of the lens proteins encountered in cataract disease is discussed.