The phase behavior of protein solutions is important for numerous phenomena in biology and soft matter. We report a lower critical solution temperature (LCST) phase behavior of aqueous solutions of a globular protein induced by multivalent metal ions around physiological temperatures. The LCST behavior manifests itself via a liquid-liquid phase separation of the protein-salt solution upon heating. Isothermal titration calorimetry and zeta-potential measurements indicate that here cation-protein binding is an endothermic, entropy-driven process. We offer a mechanistic explanation of the LCST. First, cations bind to protein surface groups driven by entropy changes of hydration water. Second, the bound cations bridge to other protein molecules, inducing an entropy-driven attraction causing the LCST. Our findings have general implications for condensation, LCST, and hydration behavior of (bio)polymer solutions as well as the understanding of biological effects of (heavy) metal ions and their hydration.

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 HoCl 3 , LaCl 3 and YCl 3 . Using UV-Vis spectroscopy and small-angle X-ray scattering (SAXS), we find that the interprotein attraction induced by Ho 3+ is very strong, while the one induced by La 3+ is comparatively weak when comparing the data to BSA-Y 3+ 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 Ho 3+ having the highest one. We propose that a combination of different cation features such as radius, polarisability and in particular hydration effects determine the proteinprotein 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.

In this article, we have studied the influence of the isotopic composition of the solvent (HO or DO) on the effective interactions and the phase behavior of the globular protein bovine serum albumin in solution with two trivalent salts (LaCl and YCl). Protein solutions with both salts exhibit a reentrant condensation phase behavior. The condensed regime (regime II) in between two salt concentration boundaries (c* < c < c**) is significantly broadened by replacing HO with DO. Within regime II, liquid-liquid phase separation (LLPS) occurs. The samples that undergo LLPS have a lower critical solution temperature (LCST). The value of LCST decreases significantly with increasing solvent fraction of DO. The effective protein-protein interactions characterized by small-angle X-ray scattering demonstrate that although changing the solvent has negligible effects below c*, where the interactions are dominated by electrostatic repulsion, an enhanced effective attraction is observed in DO above c*, consistent with the phase behavior observed. As the LCST-LLPS is an entropy-driven phase transition, the results of this study emphasize the role of entropy in solvent isotope effects.

In this work, the effects of the two anions Cl– and NO3 – on the phase behavior of bovine serum albumin (BSA) in solution with trivalent salts are compared systematically. In the presence of trivalent metal salts, negatively charged proteins such as BSA in solution undergo a reentrant condensation (RC) phase behavior, which has been established for several proteins with chlorides of trivalent salts. Here, we show that replacing Cl– by NO3 – leads to a marked change in the phase behavior. The effect is investigated for the two different cations Y3+ and La3+. The salts are thus YCl3, Y­(NO3)3, LaCl3, and La­(NO3)3. The experimental phase behavior shows that while the chloride salts induce both liquid–liquid phase separation (LLPS) and RC, the nitrate salts also induce LLPS, but RC becomes partial with La­(NO3)3 and disappears with Y­(NO3)3. The observed phase behavior is rationalized by effective protein–protein interactions which are characterized using small-angle X-ray scattering. The results based on the reduced second virial coefficients B 2/B 2 HS and 1/I(q → 0) demonstrate that the NO3 – salts induce a stronger attraction than the Cl– salts. Overall, the effective attraction, the width of the condensed regime in the RC phase diagram, and the nature of LLPS follow the order LaCl3 < YCl3 < La­(NO3)3 < Y­(NO3)3. Despite the decisive role of cations in RC phase behavior, isothermal titration calorimetry measurements indicate that replacing anions does not significantly influence the cation binding to proteins. The experimental results observed are discussed based on an “enhanced Hofmeister effect” including electrostatic and hydrophobic interactions between protein–cation complexes.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin–echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Ions are ubiquitous in nature. They play a key role for many biological processes on the molecular scale, from molecular interactions, to mechanical properties, to folding, to self‐organisation and assembly, to reaction equilibria, to signalling, to energy and material transport, to recognition etc. Going beyond monovalent ions to multivalent ions, the effects of the ions are frequently not only stronger (due to the obviously higher charge), but qualitatively different. A typical example is the process of binding of multivalent ions, such as Ca2+, to a macromolecule and the consequences of this ion binding such as compaction, collapse, potential charge inversion and precipitation of the macromolecule. Here we review these effects and phenomena induced by multivalent ions for biological (macro)molecules, from the “atomistic/molecular” local picture of (potentially specific) interactions to the more global picture of phase behaviour including, e. g., crystallisation, phase separation, oligomerisation etc. Rather than attempting an encyclopedic list of systems, we rather aim for an embracing discussion using typical case studies. We try to cover predominantly three main classes: proteins, nucleic acids, and amphiphilic molecules including interface effects. We do not cover in detail, but make some comparisons to, ion channels, colloidal systems, and synthetic polymers. While there are obvious differences in the behaviour of, and the relevance of multivalent ions for, the three main classes of systems, we also point out analogies. Our attempt of a comprehensive discussion is guided by the idea that there are not only important differences and specific phenomena with regard to the effects of multivalent ions on the main systems, but also important similarities. We hope to bridge physico‐chemical mechanisms, concepts of soft matter, and biological observations and connect the different communities further.

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 rst protein which will interact with e.g. an implant or stent. In this study, we focused on the inuence of salts (i.e., dierent cations (Y 3+ , La 3+ ) and anions (Cl − , I − )) on bovine serum albumin (BSA) in terms of its bulk behaviour, as well as its role of charges for the protein adsorption at the solid-liquid interface in order to understand and control the underlying molecular mechanisms and 1 interactions. This is part of our group's eort to gain a deep understanding of proteinprotein and protein-surface interactions in the presence of multivalent ions. In the bulk, we found not only multivalent cation-triggered phase transitions, but also a dependence on the anions. The induced attractive interactions were observed to increase from Cl − < NO − 3 < I − , resulting in iodide preventing re-entrant condensation and promoting liquidliquid 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 thickness. 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 (SiO 2 ), 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 ndings illustrate how carefully chosen surface properties and salts can directly steer the binding of anions and cations, which guide protein behaviour, thus paving the way for specic/triggered protein-protein, proteinsalt, and protein-surface interactions.

We study protein crystallization in solutions of human serum albumin (HSA) exhibiting a metastable liquid–liquid phase separation (LLPS) in the presence of trivalent salts. Specifically, we focus on the effects of dense liquid phases (DLPs) on the crystallization pathways. On the basis of the phase diagram, we choose two conditions around the LLPS binodal: one condition is located close to, but outside the LLPS region, resulting in protein clusters, but no macroscopic LLPS. Yet, a surface-enhanced unstable DLP layer is observed at the surface of the cuvette (wetting). The second condition, inside the LLPS binodal, leads to a macroscopic metastable DLP. The crystallization is followed by optical microscopy and small-angle X-ray and neutron scattering (SAXS/SANS) as well as by ultraviolet–visible spectroscopy to explore the role of LLPS. In no case evidence of nucleation inside the DLP is observed. SAXS and SANS show a monotonous growth of the crystals and a decrease of the overall material in the sample. We thus conclude that the existence of a metastable LLPS is not a sufficient condition for a two-step nucleation process. The DLP serves as a reservoir and crystal growth can be described by the Bergeron process, i.e., crystals grow directly into the dilute phase at the expense of the DLP. Furthermore, the crystallographic analysis of the resulting crystals shows that crystals with different morphology grown under different conditions share a similar crystal structure and that the metal ions create two bridging contacts within the unit cell and stabilize it.