Phase-Separation Kinetics in Protein-Salt Mixtures with Compositionally Tuned Interactions

Liquid-liquid phase separation (LLPS) in protein systems is relevant for many phenomena, from protein condensation diseases to subcellular organization to possible pathways toward protein crystallization. Understanding and controlling LLPS in proteins is therefore highly relevant for various areas of (biological) soft matter research. Solutions of the protein bovine serum albumin (BSA) have been shown to have a lower critical solution temperature-LLPS (LCST-LLPS) induceable by multivalent salts. Importantly, the nature of the multivalent cation used influences the LCST-LLPS in such systems. Here, we present a systematic ultrasmall-angle X-ray scattering investigation of the kinetics of LCST-LLPS of BSA in the presence of different mixtures of HoCl 3 and LaCl 3 , resulting in different effective interprotein attraction strengths. We monitor the characteristic length scales ξ(t, T fin ) after inducing LLPS by subjecting the respective systems to temperature jumps in their liquid-liquid coexistence regions. With increasing interprotein attraction and increasing T fin , we observe an increasing deviation from the growth law of ξ ∼ t 1/3 and an increased trend toward arrest. We thus establish a multidimensional method to tune phase transitions in our systems. Our findings help shed light on general questions regarding LLPS and the tunability of its kinetics in both proteins and colloidal systems

Packing and dynamics of a protein solution approaching the jammed state

The packing of proteins and their collective behavior in crowded media is crucial for the understanding of biological processes. Here we study the structural and dynamical evolution of solutions of the globular protein bovine serum albumin with increasing concentration via drying using small angle X-ray scattering and dynamic light scattering. We probe an evolving correlation peak on the scattering profile, corresponding to the inter-protein distance, ξ, which decreases following a power law of the protein volume fraction, ϕ. The rate of decrease in ξ becomes faster above a protein concentration of ∼200 mg ml-1 (ϕ = 0.15). The power law exponent changes from 0.33, which is typical of colloidal or protein solutions, to 0.41. During the entire drying process, we observe the development and the growth of two-step relaxation dynamics with increasing ϕ as revealed by dynamic light scattering. We find three different regimes of the dependence of ξ as a function of ϕ. In the dilute regime (ϕ 0.47), the system reaches a jammed state. Subsequently, the secondary relaxation time in this state becomes extremely slow. In this state, the protein molecules have approximately one hydration layer. This study contributes to the understanding of protein molecular packing in crowded environments and the phenomenon of density-driven jamming for soft matter systems. This journal i

Tuning phase transitions of aqueous protein solutions by multivalent cations

In the presence of trivalent cations, negatively charged globular proteins show a rich phase behaviour including reentrant condensation, crystallisation, clustering and lower critical solution temperature metastable liquid-liquid phase separation (LCST-LLPS). Here, we present a systematic study on how different multivalent cations can be employed to tune the interactions and the associated phase behaviour of proteins. We focus our investigations on the protein bovine serum albumin (BSA) in the presence of HoCl3, LaCl3 and YCl3. Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho3+ is very strong, while the one induced by La3+ is comparatively weak when comparing the data to BSA-Y3+ systems based on our previous work. Using zeta potential and isothermal titration calorimetry (ITC) measurements, we establish different binding affinities of cations to BSA with Ho3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the protein-protein interaction induced by these cations. Our findings imply that subtle differences in cation properties can be a sensitive tool to fine-tune protein-protein interactions and phase behaviour in solution

Bulk phase behavior vs interface adsorption: Specific multivalent cation and anion effects on BSA interactions

Proteins are ubiquitous and play a critical role in many areas from living organisms to protein microchips. In humans, serum albumin has a prominent role in the foreign body response since it is the first protein which will interact with, e.g., an implant or stent. In this study, we focused on the influence of salts (i.e., different cations (Y3+, La3+) and anions (Cl–, I–) on bovine serum albumin (BSA) in terms of its bulk behavior as well as the role of charges for protein adsorption at the solid–liquid interface in order to understand and control the underlying molecular mechanisms and interactions. This is part of our group’s effort to gain a deeper understanding of protein–protein and protein–surface interactions in the presence of multivalent ions. In the bulk, we established two new phase diagrams and found not only multivalent cation-triggered phase transitions, but also a dependence of the protein behavior on the type of anion. The attractive interactions between proteins were observed to increase from Cl– < NO3– < I–, resulting in iodide preventing re-entrant condensation and promoting liquid–liquid phase separation in bulk. Using ellipsometry and a quartz-crystal microbalance with dissipation (QCM-D), we obtained insight into the growth of the protein adsorption layer. Importantly, we found that phase transitions at the substrate can be triggered by certain interface properties, whether they exist in the bulk solution or not. Through the use of a hydrophilic, negatively charged surface (native silica), the direct binding of anions to the interface was prevented. Interestingly, this led to re-entrant adsorption even in the absence of re-entrant condensation in bulk. However, the overall amount of adsorbed protein was enhanced through stronger attractive protein–protein interactions in the presence of iodide salts. These findings illustrate how carefully chosen surface properties and salts can directly steer the binding of anions and cations, which guide protein behavior, thus paving the way for specific/triggered protein–protein, protein–salt, and protein–surface interactions

Interplay between Glass Formation and Liquid-Liquid Phase Separation Revealed by the Scattering Invariant

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-YCl3 system, whereas it follows the equilibrium binodal for the γ-globulin-PEG system