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The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid β, can interact with lipids, and this interaction can strongly favor the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to vesicles formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the lipid vesicles do not simply act as a surface that catalyses the nucleation reaction, but that lipid molecules take an active role in the reaction. The binding of α-synuclein to the lipid vesicles immediately induces a major structural change in the lipid assembly, which leads to a break-up into small, cylindrical and disc-like lipid-protein particles. This transition can be largely reversed by temperature changes or proteolytic protein removal. Incubation of these small, cylindrical and disc-like lipid-α-synuclein particles for several hours, however, yields amyloid fibril formation, whereby the lipids are incorporated into the fibrils.

Antibody therapies are typically based on high-concentration formulations that need to be administered subcutaneously. These conditions induce several challenges, inter alia a viscosity suitable for injection, sufficient solution stability, and preservation of molecular function. To obtain systematic insights into the molecular factors, we study the dynamics on the molecular level under strongly varying solution conditions. In particular, we use solutions of antibodies with poly(ethylene glycol), in which simple cooling from room temperature to freezing temperatures induces a transition from a well-dispersed solution into a phase-separated and macroscopically arrested system. Using quasi-elastic neutron scattering during in situ cooling ramps and in prethermalized measurements, we observe a strong decrease in antibody diffusion, while internal flexibility persists to a significant degree, thus ensuring the movement necessary for the preservation of molecular function. These results are relevant for a more dynamic understanding of antibodies in high-concentration formulations, which affects the formation of transient clusters governing the solution viscosity.

Diffusion of proteins on length scales of their size is crucial for understanding the machinery of living cells. X-ray photon correlation spectroscopy (XPCS) is currently the only way to access long-time collective diffusion on these length scales, but radiation damage so far limits the use in biological systems. We apply a new approach to use XPCS to measure cage relaxation in crowded α-crystallin solutions. This allows us to correct for radiation effects, obtain missing information on long time diffusion, and support the fundamental analogy between protein and colloid dynamical arrest.

Elastin-like peptides (ELPs) are artificially derived intrinsically disordered proteins (IDPs) mimicking the hydrophobic repeat unit in the protein elastin. ELPs are characterized by a lower critical solution temperature (LCST) in aqueous media. Here, we investigate the sequence GVG(VPGVG)3over a wide range of temperatures (below, around, and above the LCST) and peptide concentrations employing all-atom molecular dynamics simulations, where we focus on the role of intra- and inter-peptide interactions. We begin by investigating the structural properties of a single peptide that demonstrates a hydrophobic collapse with temperature, albeit moderate, as the sequence length is short. We observe a change in the interaction between two peptides from repulsive to attractive with temperature by evaluating the potential of mean force, indicating an LCST-like behaviour. Next, we explore dynamical and structural properties of peptides in multi-chain systems. We report the formation of dynamical aggregates with coil-like conformation, in which Val central residues play an important role. Moreover, the lifetime of contacts between chains strongly depends on the temperature and can be described by a power-law decay that is consistent with the LCST-like behaviour. Finally, the peptide translational and internal motion are slowed down by an increase in the peptide concentration and temperature.

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., Ions play an important role for a large variety of biological processes. Compared to monovalent ions, the effects of multivalent ions are often qualitatively different and can include complex phenomena such as charge inversion and precipitation of macromolecules. In this article, we review the effects of multivalent ions on biological (macro)molecules from the molecular to the global level. Our hope is to bridge physico‐chemical, soft matter and biological aspects, thereby stimulating discussions between these scientific communities.

Protein denaturation in concentrated solutions consists of the unfolding of the native protein structure, and subsequent cross-linking into clusters or gel networks. While the kinetic evolution of structure has been studied for some cases, the underlying microscopic dynamics of proteins has so far been neglected. However, protein dynamics is essential to understand the specific nature of assembly processes, such as diffusion-limited growth, or vitrification of dense liquids. Here, we present a study on thermal denaturation of concentrated solutions of bovine serum albumin (BSA) in D2O with and without NaCl. Using small-angle scattering, we provide information on structure before, during and after denaturation. Using quasi-elastic neutron scattering, we monitor in real-time the microscopic dynamics and dynamical confinement throughout the entire denaturation process covering protein unfolding and cross-linking. After denaturation, the protein dynamics is slowed down in salty solutions compared to those in pure water, while the stability and dynamics of the native solution appears unaffected by salt. The approach presented here opens opportunities to link microscopic dynamics to emerging structural properties, with implications for assembly processes in soft and biological matter.

We construct a coarse-grained, structure-based, low-resolution, 6-bead flexible model of bovine serum albumin (BSA, PDB: 4F5S), which is a popular example of a globular protein in biophysical research. The model is obtained via direct Boltzmann inversion using all-atom simulations of a single molecule, and its particular form is selected from a large pool of 6-bead coarse-grained models using two suitable metrics that quantify the agreement in the distribution of collective coordinates between all-atom and coarse-grained Brownian dynamics simulations of solutions in the dilute limit. For immunoglobulin G (IgG), a similar structure-based 12-bead model has been introduced in the literature [Chaudhri et al., J. Phys. Chem. B 116, 8045 (2012)] and is employed here to compare findings for the compact BSA molecule and the more anisotropic IgG molecule. We define several modified coarse-grained models of BSA and IgG, which differ in their internal constraints and thus account for a variation of flexibility. We study denser solutions of the coarse-grained models with purely repulsive molecules (achievable by suitable salt conditions) and address the effect of packing and flexibility on dynamic and static behavior. Translational and rotational self-diffusivity is enhanced for more elastic models. Finally, we discuss a number of effective sphere sizes for the BSA molecule, which can be defined from its static and dynamic properties. Here, it is found that the effective sphere diameters lie between 4.9 and 6.1 nm, corresponding to a relative spread of about ±10% around a mean of 5.5 nm.

The formation of molecular assemblies in protein solutions is of strong interest both from a fundamental viewpoint and for biomedical applications. While ordered and desired protein assemblies are indispensable for some biological functions, undesired protein condensation can induce serious diseases. As a common cofactor, the presence of salt ions is essential for some biological processes involving proteins, and in aqueous suspensions of proteins can also give rise to complex phase diagrams including homogeneous solutions, large aggregates, and dissolution regimes. Here, we systematically study the cluster formation approaching the phase separation in aqueous solutions of the globular protein BSA as a function of temperature (T), the protein concentration (c(p)) and the concentrations of the trivalent salts YCl3 and LaCl3 (c(s)). As an important complement to structural, i.e. time-averaged, techniques we employ a dynamical technique that can detect clusters even when they are transient on the order of a few nanoseconds. By employing incoherent neutron spectroscopy, we unambiguously determine the short-time self-diffusion of the protein clusters depending on c(p), c(s) and T. We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients D. For both salts, we find a simple functional form D(c(p), c(s), T) in the parameter range explored. The calculated inter-particle attraction strength, determined from the microscopic and short-time diffusive properties of the samples, increases with salt concentration and temperature in the regime investigated and can be linked to the macroscopic behavior of the samples.

International audience; Novel cold neutron backscattering spectrometers contribute substantially to the understanding of the diffusive dynamics of proteins in dense aqueous suspensions. Such suspensions are fundamentally interesting for instance in terms of the so-called macromolecular crowding, protein cluster formation, gelation, and self-assembly. Notably, backscattering spectrometers with the highest flux can simultaneously access the center-of-mass diffusion of the proteins and the superimposed internal molecular diffusive motions. The nearly complete absence of protein-protein collisions on the accessible nanosecond observation time scale even in dense protein suspensions implies that neutron backscattering accesses the so-called short-time limit for the center-of-mass diffusion. This limit is particularly interesting in terms of a theoretical understanding by concepts from colloid physics. Here we briefly review recent progress in studying protein dynamics achieved with the latest generation of backscattering spectrometers. We illustrate this progress by the first data from a protein solution using the backscattering-and-time-of-flight option BATS on IN16B at the ILL and we outline future perspectives.

Modeling diffusion of nonspherical particles presents an unsolved and considerable challenge, despite its importance for the understanding of crowding effects in biology, food technology and formulation science. A common approach in experiment and simulation is to map nonspherical objects on effective spheres to subsequently use the established predictions for spheres to approximate phenomena for nonspherical particles. Using numerical evaluation of the hydrodynamic mobility tensor, we show that this so-called effective sphere model fundamentally fails to represent the self-diffusion in solutions of ellipsoids as well as rod-like assemblies of spherical beads. The effective sphere model drastically overestimates the slowing down of self-diffusion down to volume fractions below 0.01. Furthermore, even the linear term relevant at lower volume fraction is inaccurate, linked to a fundamental misconception of effective sphere models. To overcome the severe problems related with the use of effective sphere models, we suggest a protocol to predict the short-time self-diffusion of rod-like systems, based on simulations with hydrodynamic interactions that become feasible even for more complex molecules as the essential observable shows a negligible system-size effect.

With continuously improving signal-to-noise ratios, a statistically sound analysis of quasi-elastic neutron scattering (QENS) spectra requires to fit increasingly complex models which poses several challenges. Simultaneous fits of the spectra for all recorded values of the momentum transfer become a standard approach. Spectrometers at spallation sources can have a complicated non-Gaussian resolution function which has to be described most accurately. At the same time, to speed up the fitting, an analytical convolution with this resolution function is of interest. Here, we discuss basic concepts to efficient approaches for fits of QENS spectra based on standard MATLAB and Python fit algorithms. We illustrate the fits with example data from IN16B, BASIS, and BATS.

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 redu...

International audience; 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.

We investigate the concentration-controlled formation of clusters in β-lactoglobulin (BLG) protein solutions combining structural and dynamical scattering techniques. The static structure factor from small-angle X-ray scattering as well as de-Gennes narrowing in the nanosecond diffusion function D(q) from neutron spin echo spectroscopy support a picture of cluster formation. Using neutron backscattering spectroscopy, a monotonous increase of the average hydrodynamic cluster radius is monitored over a broad protein concentration range, corresponding to oligomeric structures of BLG ranging from the native dimers up to roughly four dimers. The results suggest that BLG forms compact clusters that are static on the observation time scale of several nanoseconds. The presented analysis provides a general framework to access the structure and dynamics of macromolecular assemblies in solution.

In this article, we have studied the influence of the isotopic composition of the solvent (H2O or D2O) on the effective interactions and the phase behavior of the globular protein bovine serum albumin in solution with two trivalent salts (LaCl3 and YCl3). Protein solutions with both salts exhibit a reentrant condensation phase behavior. The condensed regime (regime II) in between two salt concentration boundaries (c* < cs < c**) is significantly broadened by replacing H2O with D2O. 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 D2O. 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 D2O above c*, consisten...

The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time- and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.

The effect of a nonspherical particle shape on the dynamics in crowded solutions presents a significant challenge for a comprehensive understanding of interaction and structural relaxation in biological and soft matter. We report that small deviations from a spherical shape induce a nonmonotonic contribution to the crowding effect on the short-time cage diffusion compared with spherical systems, using molecular dynamics simulations with mesoscale hydrodynamics of a multiparticle collision dynamics fluid in semidilute systems with volume fractions smaller than 0.35. We show that the nonmonotonic effect due to anisotropy is caused by the combination of a reduced relative mobility over the entire concentration range and a looser and less homogeneous cage packing of nonspherical particles. Our finding stresses that nonsphericity induces new complexity, which cannot be accounted for in effective sphere models, and is of great interest in applications such as formulations as well as for the fundamental understanding of soft matter in general and crowding effects in living cells in particular.

Using dynamic Monte Carlo and Brownian dynamics, we investigate a floating bond model in which particles can bind through mobile bonds. The maximum number of bonds (here fixed to 4) can be tuned by appropriately choosing the repulsive, nonadditive interactions among bonds and particles. We compute the static and dynamic structure factor (intermediate scattering function) in the vicinity of the gas-liquid critical point. The static structure exhibits a weak tetrahedral network character. The intermediate scattering function shows a temporal decay deviating from a single exponential, which can be described by a double exponential decay where the two time scales differ approximately by one order of magnitude. This time scale separation is robust over a range of wave numbers. The analysis of clusters in real space indicates the formation of noncompact clusters and shows a considerable stretch in the instantaneous size distribution when approaching the critical point. The average time evolution of the largest subcluster of given initial clusters with 10 or more particles also shows a double exponential decay. The observation of two time scales in the intermediate scattering function at low packing fractions is consistent with similar findings in globular protein solutions with trivalent metal ions that act as bonds between proteins.

Protein diffusion is not only an important process ensuring biological function but can also be used as a probe to obtain information on structural properties of protein assemblies in liquid solutions. Here, we explore the oligomerization state of ovalbumin at high protein concentrations by means of its short-time self-diffusion. We employ high-resolution incoherent quasielastic neutron scattering to access the self-diffusion on nanosecond timescales, on which interparticle contacts are not altered. Our results indicate that ovalbumin in aqueous (D2O) solutions occurs in increasingly large assemblies of its monomeric subunits with rising protein concentration. It changes from nearly monomeric toward dimeric and ultimately larger than tetrameric complexes. Simultaneously, we access information on the internal molecular mobility of ovalbumin on the nanometer length scale and compare it with results obtained for bovine serum albumin, immunoglobulin, and β-lactoglobulin.

Interactions and phase behavior of γ-globulins are of fundamental interest in biophysical and pharmaceutical research, as these are among the most abundant proteins in blood plasma. In this work, we report the characterization of the oligomeric state of bovine γ-globulin, the effective protein-protein interactions, and the colloidal stability in aqueous solution as a function of protein concentration and ionic strength. Classical biochemical techniques, such as size exclusion chromatography (SEC) and gel electrophoresis, together with small-angle X-ray and neutron scattering (SAXS/SANS), were employed for this study. The results show that bovine γ-globulin solutions are dominated by monomer and idiotype anti-idiotype dimer. Despite the flexibility and highly nonspherical shape of the protein, a simple model with a disk-type form factor and a structure factor of a square-well potential provide a satisfying description of the scattering data. The overall interactions are attractive and the strength decreases with increasing protein concentration, or adding buffer or salts. For higher protein volume fraction (>7%), the model would imply a strong particle-particle correlation which does not appear in the experimental data. This mismatch is most likely due to the smearing effect of the conformation change of proteins in solution. The stability of γ-globulin solutions is highly sensitive to protein concentration, ionic strength, and the type of added salts, such as NaCl, Na2SO4, and NaSCN. For solutions below 50 mg/mL and at low ionic strengths (1 M), typical salting-in (with NaSCN) and salting-out effects (with NaCl and Na2SO4) are observed. Results are further discussed in comparison with current studies in the literature on monoclonal antibodies.

International audience; The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals

We have studied oligo(ethylene glycol) (OEG) thiol self-assembled monolayer (SAM) coated gold nanoparticles (AuOEG) and their interactions with proteins in solutions using electrophoretic and dynamic light scattering (ELS and DLS). The results are compared with poly(ethylene glycol) (PEG) thiol coated AuNPs (AuPEG). We show that both AuOEG and AuPEG particles carry a low net negative charge and are very stable (remaining so for more than one year), but long-term aging or dialysis can reduce the stability. If the decorated AuNPs are mixed with bovine serum albumin (BSA), both effective size and zeta-potential of the AuNPs remain unchanged, indicating no adsorption of BSA to the colloid surface. However, when mixed with lysozyme, zeta-potential values increase with protein concentrations and lead to a charge inversion, indicating adsorption of lysozyme to the colloid surface. The colloidal solutions of AuOEG become unstable near zero charge, indicated by a cluster peak in the DLS measurements. The AuPEG solutions show similar charge inversion upon addition of lysozyme, but the solutions are stable under all experimental conditions, presumably because of the strong steric effect of PEG. Washing the protein bound colloids by centrifugation can remove only part of the adsorbed lysozyme molecules indicating that a few proteins adsorb strongly to the colloids. The effective charge inversion and rather strongly bound lysozyme on the colloid surface may suggest that in addition to the charges formed at the SAM-water interface, there are defects on the surface of the colloid, which are accessible to the proteins. The results of this study of surface charge, and stability shed light on the interaction with proteins of SAM coated AuNPs and their applications.

Tuning of protein surface charge is a fundamental mechanism in biological systems. Protein charge is regulated in a physiological context by pH and interaction with counterions. We report on charge inversion and the related reentrant condensation in solutions of globular proteins with different multivalent metal cations. In particular, we focus on the changes in phase behavior and charge regulation due to pH effects caused by hydrolysis of metal ions. For several proteins and metal salts, charge inversion as measured by electrophoretic light scattering is found to be a universal phenomenon, the extent of which is dependent on the specific protein-salt combination. Reentrant phase diagrams show a much narrower phase-separated regime for acidic salts such as AlCl3 and FeCl3 compared to neutral salts such as YCl3 or LaCl3 . The differences between acidic and neutral salts can be explained by the interplay of pH effects and binding of the multivalent counterions. The experimental findings are reproduced with good agreement by an analytical model for protein charging taking into account ion condensation, metal ion hydrolysis and interaction with charged amino acid side chains on the protein surface. Finally, the relationship of charge inversion and reentrant condensation is discussed, suggesting that pH variation in combination with multivalent cations provides control over both attractive and repulsive interactions between proteins.

During protein crystallization and purification, proteins are commonly found in concentrated salt solutions. The exact interplay of the hydration shell, the salt ions, and protein-protein interactions under these conditions is far from being understood on a fundamental level, despite the obvious practical relevance. We have studied a model globular protein (bovine serum albumin, BSA) in concentrated salt solutions by small-angle neutron scattering (SANS). The data are also compared to previous studies using SAXS. The SANS results for dilute protein solutions give an averaged volume of BSA of 91700 3, which is about 37% smaller than that determined by SAXS. The difference in volume corresponds to the contribution of a hydration shell with a hydration level of 0.30 g g -1 protein. The forward intensity I(0) determined from Guinier analysis is used to determine the second virial coefficient, A 2, which describes the overall protein interactions in solution. It is found that A 2 follows the reverse order of the Hofmeister series, i.e. (NH 4) 2SO 4 < Na 2SO 4 < NaOAc < NaCl < NaNO 3 < NaSCN. The dimensionless second virial coefficient B 2, corrected for the particle volume and molecular weight, has been calculated using different approaches, and shows that B 2 with corrections for hydration and the non-spherical shape of the protein describes the interactions better than those determined from the bare protein. SANS data are further analyzed in the full q-range using liquid theoretical approaches, which gives results consistent with the A 2 analysis and the experimental structure factor. © 2012 the Owner Societies.

We briefly summarize the recent progress in tuning protein interactions as well as phase behavior in protein solutions using multivalent metal ions. We focus on the influence of control parameters and the mechanism of reentrant condensation, the metastable liquid–liquid phase separation and classical vs. non-classical pathways of protein crystallization.

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.

We report on a joint experimental–theoretical study of collective diffusion in, and static shear viscosity of solutions of bovine serum albumin (BSA) proteins, focusing on the dependence on protein and salt concentration. Data obtained from dynamic light scattering and rheometric measurements are compared to theoretical calculations based on an analytically treatable spheroid model of BSA with isotropic screened Coulomb plus hard-sphere interactions. The only input to the dynamics calculations is the static structure factor obtained from a consistent theoretical fit to a concentration series of small-angle X-ray scattering (SAXS) data. This fit is based on an integral equation scheme that combines high accuracy with low computational cost. All experimentally probed dynamic and static properties are reproduced theoretically with an at least semi-quantitative accuracy. For lower protein concentration and low salinity, both theory and experiment show a maximum in the reduced viscosity, caused by the electrostatic repulsion of proteins. On employing our theoretical and experimental results, the applicability range of a generalized Stokes–Einstein (GSE) relation connecting viscosity, collective diffusion coefficient, and osmotic compressibility, proposed by Kholodenko and Douglas [Phys. Rev. E, 1995, 51, 1081] is examined. Significant violation of the GSE relation is found, both in experimental data and in theoretical models, in concentrated systems at physiological salinity, and under low-salt conditions for arbitrary protein concentrations.

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.

We have studied nanoscale diffusion of membrane hydration water in fluid-phase lipid bilayers made of 1,2-dimyristoyl-3-phosphocholine (DMPC) using incoherent quasi-elastic neutron scattering. Dynamics were fit directly in the energy domain using the Fourier transform of a stretched exponential. By using large, 2-dimensional detectors, lateral motions of water molecules and motions perpendicular to the membranes could be studied simultaneously, resulting in 2-dimensional maps of relaxation time, τ, and stretching exponent, β. We present experimental evidence for anomalous (sub-diffusive) and anisotropic diffusion of membrane hydration water molecules over nanometer distances. By combining molecular dynamics and Brownian dynamics simulations, the potential microscopic origins for the anomaly and anisotropy of hydration water were investigated. Bulk water was found to show intrinsic sub-diffusive motion at time scales of several picoseconds, likely related to caging effects. In membrane hydration water, however, the anisotropy of confinement and local dynamical environments leads to an anisotropy of relaxation times and stretched exponents, indicative of anomalous dynamics.

We report a real-time study on protein crystallization in the presence of multivalent salts using small angle X-ray scattering (SAXS) and optical microscopy, focusing particularly on the nucleation mechanism as well as on the role of the metastable intermediate phase (MIP). Using bovine beta-lactoglobulin as a model system in the presence of the divalent salt CdCl2, we have monitored the early stage of crystallization kinetics which demonstrates a two-step nucleation mechanism: protein aggregates form a MIP, which is followed by the nucleation of crystals within the MIP. Here we focus on characterizing and tuning the structure of the MIP using salt and the related effects on the two-step nucleation kinetics. The results suggest that increasing the salt concentration near the transition zonepseudo-c** enhances the energy barrier for both MIPs and crystal nucleation, leading to slow growth. The structural evolution of the MIP and its effect on subsequent nucleation is discussed based on the growth kinetics. The observed kinetics can be well described, using a rate-equation model based on a clear physical two-step picture. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization, but also elucidates the role and the structural signature of the MIPs in the nonclassical process of protein crystallization.

We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.

The short-time self-diffusion D of the globular model protein bovine serum albumin in aqueous (D2O) solutions has been measured comprehensively as a function of the protein and trivalent salt (YCl3) concentration, noted cp and cs, respectively. We observe that D follows a universal master curve D(cs,cp) = D(cs = 0,cp) g(cs/cp), where D(cs = 0,cp) is the diffusion coefficient in the absence of salt and g(cs/cp) is a scalar function solely depending on the ratio of the salt and protein concentration. This observation is consistent with a universal scaling of the bonding probability in a picture of cluster formation of patchy particles. The finding corroborates the predictive power of the description of proteins as colloids with distinct attractive ion-activated surface patches.

Elastic fixed window scans of incoherent neutron scattering are an established and frequently employed method to study dynamical changes, usually over a broad temperature range or during a process such as a conformational change in the sample. In particular, the apparent mean-squared displacement can be extracted via a model-free analysis based on a solid physical interpretation as an effective amplitude of molecular motions. Here, we provide a new account of elastic and inelastic fixed window scans, defining a generalized mean-squared displacement for all fixed energy transfers. We show that this generalized mean-squared displacement in principle contains all information on the real mean-square displacement accessible in the instrumental time window. The derived formula provides a clear understanding of the effects of instrumental resolution on the apparent mean-squared displacement. Finally, we show that the generalized mean-square displacement can be used as a model-free indicator on confinement effects within the instrumental time window.

The dynamics of proteins in solution is a complex and hierarchical process, affected by the aqueous environment as well as temperature. We present a comprehensive study on nanosecond time and nanometer length scales below, at, and above the denaturation temperature Td. Our experimental data evidence dynamical processes in protein solutions on three distinct time scales. We suggest a consistent physical picture of hierarchical protein dynamics: (i) self-diffusion of the entire protein molecule is confirmed to agree with colloid theory for all temperatures where the protein is in its native conformational state. At higher temperatures T > Td, the self-diffusion is strongly obstructed by cross-linking or entanglement. (ii) The amplitude of backbone fluctuations grows with increasing T, and a transition in its dynamics is observed above Td. (iii) The number of mobile side-chains increases sharply at Td while their average dynamics exhibits only little variations. The combination of quasi-elastic neutron scattering and the presented analytical framework provides a detailed microscopic picture of the protein molecular dynamics in solution, thereby reflecting the changes of macroscopic properties such as cluster formation and gelation.

The understanding of protein interactions to control phase and nucleation behavior of protein solutions is an important challenge for soft matter, biological and medical research. Here, we present ion bridges of multivalent cations between proteins as an ion-activated mechanism for patchy interaction that is directly supported by experimental findings in protein crystals. A deep understanding of experimentally observed phenomena in protein solutions--including charge reversal, reentrant condensation, metastable liquid-liquid phase separation, cluster formation and different pathways of crystallization--is gained by an analytic model that directly displays parameter dependencies and physical connections. The direct connection between experiment and theory provides a conceptual framework for future experimental, computational and theoretical research on topics such as rational design of phase behavior and crystallization pathways on the basis of the statistical physics of patchy particles.

The phase behavior of protein solutions is affected by additives such as crowder molecules or salts. In particular, upon addition of multivalent counterions, a reentrant condensation can occur; i.e., protein solutions are stable for low and high multivalent ion concentrations but aggregating at intermediate salt concentrations. The addition of monovalent ions shifts the phase boundaries to higher multivalent ion concentrations. This effect is found to be reflected in the protein interactions, as accessed via small-angle X-ray scattering. Two simulation schemes (a Monte Carlo sampling of the counterion binding configurations using the detailed protein structure and an analytical coarse-grained binding model) reproduce the shifts of the experimental phase boundaries. The results support a consistent picture of the protein interactions responsible for the phase behavior. The repulsive Coulomb interaction is varied by the binding of multivalent counterions and additionally screened by any increase of the ionic strength. The attractive interaction is induced by the binding of multivalent ions, most likely due to ion bridging between protein molecules. The overall picture of these competing interactions provides interesting insight into possible mechanisms for tailoring interactions in solutions via salt effects.

Dynamics in protein solutions is essential for both protein function and cellular processes. The hierarchical complexity of global protein diffusion, side-chain diffusion, and microscopic motions of chemical groups renders a complete understanding challenging. We present results from quasi-elastic neutron scattering on protein solutions of γ-globulin over a wide range of volume fractions. Translational and rotational diffusion can be self-consistently separated from internal motions. The global diffusion is consistent with predictions for effective spheres even though the branched molecular shape differs considerably from a colloidal sphere. The internal motions are characterized both geometrically and dynamically, suggesting a picture of methyl rotations and restricted diffusion of side chains. We show that the advent of new neutron spectrometers allows the study of current questions including the coupling of intracellular dynamics and protein function.

The formation of protein clusters as precursors for crystallization and phase separation is of fundamental and practical interest in protein science. Using multivalent ions, the strengths of both long-range Coulomb repulsion and short-range attraction can be tuned in protein solutions, representing a well-controlled model system to study static and dynamic properties of clustering during the transition from a charge-stabilized to an aggregate regime. Here, we study compressibility, diffusion, and size of solutes by means of static (SLS) and dynamic light scattering (DLS) in solutions of bovine serum albumin (BSA) and YCl3. For this and comparable systems, an increasing screening and ultimately inversion of the protein surface charge induce a rich phase behavior including reentrant condensation, liquid-liquid phase separation and crystallization, which puts the cluster formation in the context of precursor formation and nucleation of liquid and crystalline phases. We find that, approaching the turbid aggregate regime with increasing salt concentration cs, the diffusion coefficients decrease and the scattered intensity increases by orders of magnitude, evidencing increasing correlation lengths likely associated with clustering. The combination of static and dynamic observations suggests the formation of BSA clusters with a size on the order of 100 nm. The global thermodynamic state seems to be stable over at least several hours. Surprisingly, results on collective diffusion and inverse compressibility from different protein concentrations can be rescaled into master curves as a function of cs/c*, where c* is the critical salt concentration of the transition to the turbid aggregate regime.

We present an experimental study combined with a theoretical discussion of the effective interactions in protein solutions approaching a liquid-liquid phase separation (LLPS) induced by addition of multivalent metal ions. The reduced second virial coefficient, B 2/B 2 HS, is used to describe the interaction and discussed with theoretical predictions for colloidal systems. We have determined the salt and protein partitioning in the two coexisting phases, which provides the isothermal binodal of the LLPS in the (cp, cs ) plane. Two sets of samples, away from and at the LLPS binodal were measured by static light scattering (SLS) and small angle X-ray scattering (SAXS) to determine the second virial coefficient. In all cases, B 2/B 2 HS is negative in the condensed regime, and increases by approaching the upper critical point in the (cp, cs ) plane. The results are compared with a simple colloidal model with isotropic short-ranged attraction and a thermodynamic criterion based on the reduced second virial coefficient. We discuss the application of this theoretical prediction to interpret experimental observations. © 2014 Elsevier B.V. All rights reserved.

We discuss the phase behavior and in particular crystallization of a model globular protein (beta-lactoglobulin) in solution in the presence of multivalent electrolytes. It has been shown previously that negatively charged globular proteins at neutral pH in the presence of multivalent counterions undergo a "re-entrant condensation (RC)" phase behavior (Zhang et al., Phys. Rev. Lett., 2008, 101, 148101), i.e. a phase-separated regime occurs in between two critical salt concentrations, c* < c**, giving a metastable liquid-liquid phase separation (LLPS). Crystallization from the condensed regime has been observed to follow different mechanisms. Near c*, crystals grow following a classic nucleation and growth mechanism; near c**, the crystallization follows a two-step crystallization mechanism, i.e, crystal growth follows a metastable LLPS. In this paper, we focus on the two-step crystal growth near c**. SAXS measurements indicate that proteins form clusters in this regime and the cluster size increases approaching c**. Upon lowering the temperature, in situ SAXS studies indicate that the clusters can directly form both a dense liquid phase and protein crystals. During the crystal growth, the metastable dense liquid phase is dissolved. Based on our observations, we discuss a nucleation mechanism starting from clusters in the dilute phase from a metastable LLPS. These protein clusters behave as the building blocks for nucleation, while the dense phase acts as a reservoir ensuring constant protein concentration in the dilute phase during crystal growth. © The Royal Society of Chemistry 2012.

Using both quasi-elastic and fixed-window neutron spectroscopy, we study the dynamics of highly concentrated aqueous protein solutions of bovine serum albumin around the denaturing transition. For the temperature range 280 K < T < 370 K, the total mean-squared displacement 〈u 2〉 is recorded. Below and above the denaturing, we observe that 〈u 2〉 increases monotonically with T, but at the denaturing transition it decreases strongly. This observation can be rationalized and quantitatively modeled as a transition from a liquid protein solution to a gel-like state. Atomic vibrations, molecular subunit diffusion and, most importantly, diffusion of the entire protein determine 〈u 2〉. The latter is strongly hindered due to entanglement and cross-linking of the chains and causes the pronounced decrease of 〈u 2〉. Using information from the full quasi-elastic signal, we separate the diffusion contribution from 〈u 2〉 and reveal the transition temperature. For the analysis of this separation, we introduce a general concept, which is applicable to other colloid systems exhibiting both center-of-mass and internal dynamics. © 2012 The Royal Society of Chemistry.

We demonstrate that a metastable liquid-liquid phase separation (LLPS) in protein aqueous solutions can be induced by multivalent metal ions at room temperature. We determine the salt and protein partitioning in the two coexisting phases. The structure factor obtained by small angle X-ray scattering provides direct evidence for a short-ranged attraction, which leads to the metastability of the LLPS. An extended phase diagram with three control parameters (temperature, protein and salt concentration) provides a conclusive physical picture consistent with a criterion for the second virial coefficient. The presented isothermal control mechanism of the phase behavior opens new perspectives for the understanding of controlled phase behavior in nature. Furthermore, we discuss the application of this framework in predicting and optimizing conditions for protein crystallization. © 2012 The Royal Society of Chemistry.