Solutes added to buffered solutions directly impact protein folding. Protein stabilization by cosolutes or crowders has been shown to be largely driven by protein–cosolute volume exclusion complemented by chemical and soft interactions. By contrast to previous studies that indicate the invariably destabilizing role of soft protein–sugar attractions, we show here that soft interactions with sugar cosolutes are protein-specific and can be stabilizing or destabilizing. We experimentally follow the folding of two model miniproteins that are only marginally stable but in the presence of sugars and polyols fold into representative and distinct secondary structures: β-hairpin or α-helix. Our mean-field model reveals that while protein–sugar excluded volume interactions have a similar stabilizing effect on both proteins, the soft interactions add a destabilizing contribution to one miniprotein but further stabilize the other. Using molecular dynamics simulations, we link the soft protein–cosolute interactions to the weakening of direct protein–water hydrogen bonding due to the presence of sugars. Although these weakened hydrogen bonds destabilize both the native and denatured states of the two proteins, the resulting contribution to the folding free energy can be positive or negative depending on the amino acid sequence. This study indicates that the significant variation between proteins in their soft interactions with sugar determines the specific response of different proteins, even to the same sugar.

Deep eutectic solvents (DESs) show promise in pharmaceutical applications, most prominently as excellent solubilizers. Yet, because DES are complex multi-component mixtures, it is challenging to dissect the contribution of each component to solvation. Moreover, deviations from the eutectic concentration lead to phase separation of the DES, making it impractical to vary the ratios of components to potentially improve solvation. Water addition alleviates this limitation as it significantly decreases the melting temperature and stabilizes the DES single-phase region. Here, we follow the solubility of β-cyclodextrin (β-CD) in DES formed by the eutectic 2:1 mole ratio of urea and choline chloride (CC). Upon water addition to DES, we find that at almost all hydration levels, the highest β-CD solubility is achieved at DES compositions that are shifted from the 2:1 ratio. At higher urea to CC ratios, due to the limited solubility of urea, the optimum composition allowing the highest β-CD solubility is reached at the DES solubility limit. For mixtures with higher CC concentration, the composition allowing optimal solvation varies with hydration. For example, β-CD solubility at 40 wt% water is enhanced by a factor of 1.5 for a 1:2 urea to CC mole ratio compared with the 2:1 eutectic ratio. We further develop a methodology allowing us to link the preferential accumulation of urea and CC in the vicinity of β-CD to its increased solubility. The methodology we present here allows a dissection of solute interactions with DES components that is crucial for rationally developing improved drug and excipient formulations.

Designed to enhance the solubility of hardly soluble species in water, many excipient formulations for active drugs or other solutes contain two or more cosolutes. And yet, little is known about the mechanism through which excipients act in combination, and how the efficacy of each component toward drug solubility changes compared to when they are acting alone. Here we study aqueous mixtures of urea and inorganic salts and determine their efficacy to solubilize β-cyclodextrin, a cyclic carbohydrate and a key ingredient in many drug formulations. We show that the order in which the salts increased β-cyclodextrin solubility follows the Hofmeister series both in the presence and absence of urea. However, the solubility in many of the urea-salt mixtures is notably higher than the sum of the solubilities in each cosolute on its own, suggesting a synergistic effect between solutes. By determining the activity of urea and NaClO₄, the combination showing the strongest synergy, we show that their remarkable solubility enhancement at high concentration is due to a strong urea-assisted accumulation of NaClO₄ at the vicinity of β-cyclodextrin. We discuss the molecular interactions that lead to this induced accumulation of NaClO₄. Our findings provide new insight into the mechanism of solvation by multiple cosolutes and should aid in the rational design of tailored excipient formulations.

Binary compositions of surface ligands are known to improve the colloidal stability and fluorescence quantum yield of nanocrystals (NCs), due to ligand–ligand interactions and surface organization. Herein, we follow the thermodynamics of a ligand exchange reaction of CdSe NCs with alkylthiol mixtures. The effects of ligand polarity and length difference on ligand packing were investigated using isothermal titration calorimetry (ITC). The thermodynamic signature of the formation of mixed ligand shells was observed. Correlating the experimental results with thermodynamic mixing models has allowed us to calculate the interchain interactions and to infer the final ligand shell configuration. Our findings demonstrate that, in contrast to macroscopic surfaces, the small dimensions of the NCs and the subsequent increased interfacial region between dissimilar ligands allow the formation of a myriad of clustering patterns, controlled by the interligand interactions. This work provides a fundamental understanding of the parameters determining the ligand shell structure and should help guide smart surface design toward NC-based applications.

Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein–crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG–protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG–protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.

Lipid nanodiscs are nanometric bilayer patches enveloped by confining structures, commonly composed of membrane scaffolding proteins (MSPs). To resolve the interplay between MSP geometry, lipid confinement, and membrane material properties on the nanodisc shape, we apply a continuum elastic theory accounting for lipid bending, tilting, and area deformations. The equilibrium nanodisc shape is then determined by minimizing the elastic free energy functional. Analytic expressions derived under simplifying assumptions demonstrate that the nanodisc shape is sensitive to its size, lipid density, and the lipid tilt and thickness imposed at the contact with the MSP. Under matching physical parameters, these expressions quantitatively reproduce the shape of nanodiscs seen in molecular dynamics simulations, but only if lipid tilt is explicitly considered. We further demonstrate how the bending rigidity can be extracted from the membrane shape profile by fitting the numerically minimized full elastic functional to the membrane shape found in simulations. This fitting procedure faithfully informs on the bending rigidity of nanodiscs larger than ca. 5 nm in radius. The fitted profiles accurately reproduce the increase in bending modulus found using real-space fluctuation analysis of simulated nanodiscs and, for large nanodiscs, also accurately resolve its spatial variations. Our study shows how deformations in lipid patches confined in nanodiscs can be well described by a continuum elastic theory and how this fit can be used to determine local material properties from shape analysis of nanodiscs in simulations. This methodology could potentially allow direct determination of lipid properties from experiments, for example cryo-electron microscopy images of lipid nanodiscs, thereby allowing to guide the development of future nanodisc formulations with desired properties.

To cope with stress induced by high salinity and hydrostatic pressure, some marine animals accumulate small organic solutes called osmolytes. Most notable among these osmolytes are the denaturant urea, and trimethylamine N-oxide (TMAO) that is known to stabilize proteins. Although their effects on proteins and nucleic acids have been extensively studied, osmolytes are less commonly studied in the context of lipids, which are a crucial component in many cellular processes. Here we resolve the mechanism for urea’s action on the forces acting between lipid membranes, in the presence and absence of TMAO. We find that unlike the way urea denatures proteins, and by contrast to TMAO, urea does not preferentially interact with net-neutral lipid membranes. Instead, urea modulates the interactions between membranes mainly by weakening the van der Waals attraction between bilayers. Interestingly, regardless of concentration, the effects of urea and TMAO appear to be additive to a large extent, so that the presence of one osmolyte hardly changes the interaction of the other with lipid. Weak non-additive effects are likely due to structural changes in the lipid membrane induced by the osmolytes. Finally, we comment on the potential role of osmolytes acting together in the modification of lipid adhesion and fusion.

Cells are crowded, but proteins are almost always studied in dilute aqueous buffer. We review the experimental evidence that crowding affects the equilibrium thermodynamics of protein stability and protein association and discuss the theories employed to explain these observations. In doing so, we highlight differences between synthetic polymers and biologically relevant crowders. Theories based on hard-core interactions predict only crowding-induced entropic stabilization. However, experiment-based efforts conducted under physiologically relevant conditions show that crowding can destabilize proteins and their complexes. Furthermore, quantification of the temperature dependence of crowding effects produced by both large and small cosolutes, including osmolytes, sugars, synthetic polymers, and proteins, reveals enthalpic effects that stabilize or destabilize proteins. Crowding-induced destabilization and the enthalpic component point to the role of chemical interactions between and among the macromolecules, cosolutes, and water. We conclude with suggestions for future studies.

Surface ligands of semiconductor nanocrystals (NCs) play key roles in determining their colloidal stability and physicochemical properties and are thus enablers also for the NCs flexible manipulation toward numerous applications. Attention is usually paid to the ligand binding group, while the impact of the ligand chain backbone structure is less discussed. Using isothermal titration calorimetry (ITC), we studied the effect of structural changes in the ligand chain on the thermodynamics of the exchange reaction for oleate coated CdSe NCs, comparing linear and branched alkylthiols. The investigated alkylthiol ligands differed in their backbone length, branching position, and branching group length. Compared to linear ligands, lower exothermicity and entropy loss were observed for an exchange with branched ligands, due to steric hindrance in ligand packing, thereby justifying their previous classification as “entropic ligands”. Mean-field calculations for ligand binding demonstrate the contribution to the overall entropy originating from ligand conformational entropy, which is diminished upon binding mainly by packing of NC-bound ligands. Model calculations and the experimental ITC data both point to an interplay between the branching position and the backbone length in determining the entropic nature of the branched ligand. Our findings suggest that the most entropic ligand should be a short, branched ligand with short branching group located toward the middle of the ligand chain. The insights provided by this work also contribute to a future smarter NC surface design, which is an essential tool for their implementation in diverse applications.

Molecular self-assembly forms structures of well-defined organization that allow control over material properties, affording many advanced technological applications. Although the self-assembly of molecules is seemingly spontaneous, the structure into which they assemble can be altered by carefully modulating the driving forces. Here we study the self-assembly within the constraints of nanoconfined closed spherical volumes of polymeric nanocapsules, whereby a mixture of polyester-polyether block copolymer and methacrylic acid methyl methacrylate copolymer forms the entrapping capsule shell of nanometric dimensions. We follow the organization of the organic dye indigo carmine that serves as a model building unit due to its tendency to self-assemble into flat lamellar molecular sheets. Analysis of the structures formed inside the nanoconfined space using cryogenic-transmission electron microscopy (cryo-TEM) and cryogenic-electron tomography (cryo-ET) reveal that confinement drives the self-assembly to produce tubular scroll-like structures of the dye. Combined continuum theory and molecular modeling allow us to estimate the material properties of the confined nanosheets, including their elasticity and brittleness. Finally, we comment on the formation mechanism and forces that govern self-assembly under nanoconfinement.

Vesicles enriched in certain negatively charged lipids, such as phosphatidylserine and PIP2, are known to undergo fusion in the presence of calcium ions without assistance from protein assemblies. Other lipids do not exhibit this propensity, even if they are negatively charged. Using our recently developed methodology, we extract elastic properties of a representative set of lipids. This allows us to trace the origin of lipid-calcium selectivity in membrane fusion to the formation of lipid clusters with long-range correlations that induce negative curvature on the membrane surface. Furthermore, the clusters generate lateral tension in the headgroup region at the membrane surface, concomitantly also stabilizing negative Gaussian curvature. Finally, calcium binding also reduces the orientational polarization of water around the membrane head groups, potentially reducing the hydration force acting between membranes. Binding calcium only weakly increases membrane bending rigidity and tilt moduli, in agreement with experiments. We show how the combined effects of calcium binding to membranes lower the barriers along the fusion pathway that lead to the formation of the fusion stalk as well as the fusion pore.

From stem cell freeze-drying to organ storage, considerable recent efforts have been directed toward the development of new preservation technologies. A prominent protein stabilizing strategy involves vitrification in glassy matrices, most notably those formed of sugars such as the biologically relevant preservative trehalose. Here, we compare the folding thermodynamics of a model miniprotein in solution and in the glassy state of the sugars trehalose and glucose. Using synchrotron radiation circular dichroism (SRCD), we find that the same native structure persists in solution and glass. However, upon transition to the glass, a completely different, conformationally restricted unfolded state replaces the disordered denatured state found in solution, potentially inhibiting misfolding. Concomitantly, a large exothermic contribution is observed in glass, exposing the stabilizing effect of interactions with the sugar matrix on the native state. Our results shed light on the mechanism of protein stabilization in sugar glass and should aid in future preservation technologies.

By complexing with hydrophobic compounds, cyclodextrins afford increased solubility and thermodynamic stability to hardly soluble compounds, thereby underlining their invaluable applications in pharmaceutical and other industries. However, common cyclodextrins such as β-cyclodextrin, suffer from limited solubility in water, which often leads to precipitation and formation of unfavorable aggregates, driving the search for better solvents. Here, we study the solvation of cyclodextrin in deep eutectic solvents (DESs), environmentally friendly media that possess unique properties. We focus on reline, the DES formed from choline chloride and urea, and resolve the mechanism through which its constituents elevate β-cyclodextrin solubility in hydrated solutions compared to pure water or dry reline. Combining experiments and simulations, we determine that the remarkable solubilization of β-cyclodextrin in hydrated reline is mostly due to the inclusion of urea inside β-cyclodextrin’s cavity and at its exterior surfaces. The role of choline chloride in further increasing solvation is twofold. First, it increases urea’s solubility beyond the saturation limit in water, ultimately leading to much higher β-cyclodextrin solubility in hydrated reline in comparison to aqueous urea solutions. Second, choline chloride increases urea’s accumulation in β-cyclodextrin’s vicinity. Specifically, we find that the accumulation of urea becomes stronger at high reline concentrations, as the solution transitions from reline-in-water to water-in-reline, where water alone cannot be regarded as the solvent. Simulations further suggest that in dry DES, the mechanism of β-cyclodextrin solvation changes so that reline acts as a quasi-single component solvent that lacks preference for the accumulation of urea or choline chloride around β-cyclodextrin.

By complexing with hydrophobic compounds, cyclodextrins afford increased solubility and thermodynamic stability to hardly soluble compounds, thereby underlining their invaluable applications in pharmaceutical and other industries. However, common cyclodextrins such as β-cyclodextrin, suffer from limited solubility in water, which often leads to precipitation and formation of unfavorable aggregates, driving the search for better solvents. Here, we study the solvation of cyclodextrin in deep eutectic solvents (DESs), environmentally friendly media that possess unique properties. We focus on reline, the DES formed from choline chloride and urea, and resolve the mechanism through which its constituents elevate β-cyclodextrin solubility in hydrated solutions compared to pure water or dry reline. Combining experiments and simulations, we determine that the remarkable solubilization of β-cyclodextrin in hydrated reline is mostly due to the inclusion of urea inside β-cyclodextrin’s cavity and at its exterior surfaces. The role of choline chloride in further increasing solvation is twofold. First, it increases urea’s solubility beyond the saturation limit in water, ultimately leading to much higher β-cyclodextrin solubility in hydrated reline in comparison to aqueous urea solutions. Second, choline chloride increases urea’s accumulation in β-cyclodextrin’s vicinity. Specifically, we find that the accumulation of urea becomes stronger at high reline concentrations, as the solution transitions from reline-in-water to water-in-reline, where water alone cannot be regarded as the solvent. Simulations further suggest that in dry DES, the mechanism of β-cyclodextrin solvation changes so that reline acts as a quasi-single component solvent that lacks preference for the accumulation of urea or choline chloride around β-cyclodextrin.

Protein aggregation is involved in a variety of diseases, including neurodegenerative diseases and cancer. The cellular environment is crowded by a plethora of cosolutes comprising small molecules and biomacromolecules at high concentrations, which may influence the aggregation of proteins in vivo. To account for the effect of cosolutes on cancer-related protein aggregation, we studied their effect on the aggregation of the cancer-related L106R mutant of the Axin protein. Axin is a key player in the Wnt signaling pathway, and the L106R mutation in its RGS domain results in a native molten globule that tends to form native-like aggregates. This results in uncontrolled activation of the Wnt signaling pathway, leading to cancer. We monitored the aggregation process of Axin RGS L106R in vitro in the presence of a wide ensemble of cosolutes including polyols, amino acids, betaine and polyethylene glycol (PEG) crowders. Except myo-inositol, all polyols decreased RGS L106R aggregation, with carbohydrates exerting the strongest inhibition. Conversely, betaine and PEGs enhanced aggregation. These results are consistent with the reported effects of osmolytes and crowders on the stability of molten globular proteins and with both amorphous and amyloid aggregation mechanisms. We suggest a model of Axin L106R aggregation in vivo, whereby molecularly small osmolytes keep the protein as a free solublemolecule but the increased crowding of the bound state by macromolecules induces its aggregation at the nano-scale. To our knowledge, this is the first systematic study on the effect of osmolytes and crowders on a process of native-like aggregation involved in pathology, as it sheds light on the contribution of cosolutes to the onset of cancer as a protein misfolding disease, and on the relevance of aggregation in the molecular aetiology of cancer.

Ubiquitous in the cellular milieu, small organic compounds termed osmolytes help to regulate the response to environmental stress. Elasmobranchii, such as sharks, accumulate high concentrations of several osmolytes, most notably urea, trimethylamine N‐oxide (TMAO), and glycine betaine (GB). These three compounds are used to osmoregulate the organism's body fluids, so that they counteract seawater salinity, as well as the destabilizing effects of hydrostatic pressure on cells and their molecular components. Herein we focus on glycine betaine and show how it modifies interactions between lipid membranes. We find that the addition of GB exerts an apparent attractive force that draws neighboring membranes toward one another. We show that, at the molecular level, this apparent attraction between membranes in the presence of GB is due to its preferential exclusion from the space between adjacent membranes, which thereby exerts an osmotic pressure that brings membranes closer together. This action is similar to the one we have previously reported for TMAO. However, we find that the net effect of GB is significantly smaller than that of TMAO, because GB concomitantly significantly weakens van der Waals attractions between membranes by changing the dielectric properties of the intervening solution. Finally, we show how GB acts in combination with urea. At high total concentrations and over a wide range of GB‐to‐urea ratios, urea counteracts the effect of GB, so that the equilibrium separation between membranes is close to their values in pure water. This finding supports the prevailing idea that mixtures of several osmolytes can achieve minimal net impact on biomacromolecular stability while also counteracting osmotic stress.

The interaction between lipid membranes and ions is associated with a range of key physiological processes. Most earlier studies have focused on the interaction of lipids with cations, while the specific effects of the anions have been largely overlooked. Owing to dissolved atmospheric carbon dioxide, bicarbonate is an important ubiquitous anion in aqueous media. In this paper, we examined the effect of bicarbonate anions on the interactions between dipolar lipid membranes in the presence of previously adsorbed calcium cations. Using a combination of solution X-ray scattering, osmotic stress, and molecular dynamic simulations, we followed the interactions between 1,2-didodecanoyl-sn-glycero-3-phosphocholine (DLPC) lipid membranes that were dialyzed against CaCl₂ solutions in the presence and absence of bicarbonate anions. Calcium cations adsorbed onto DLPC membranes charge them and lead to their swelling. In the presence of bicarbonate anions, however, the calcium cations can tightly couple one dipolar DLPC membrane to the other and form a highly condensed and dehydrated lamellar phase with a repeat distance of 3.45±0.02 nm. Similar tight condensation and dehydration has only been observed between charged membranes in the presence of multivalent counterions. Bridging between bilayers by calcium bicarbonate complexes induced this arrangement. Furthermore, in this condensed phase the lipid molecules and the adsorbed ions were arranged in a 2D oblique lattice.

Lipid nanodiscs are small synthetic lipid bilayer structures that are stabilized in solution by special circumscribing (or scaffolding) proteins or polymers. Because they create native-like environments for transmembrane proteins, lipid nanodiscs have become a powerful tool for structural determination of this class of systems when combined with cryo-electron microscopy or nuclear magnetic resonance. The elastic properties of lipid bilayers determine how the lipid environment responds to membrane protein perturbations, and how the lipid in turn modifies the conformational state of the embedded protein. However, despite the abundant use of nanodiscs in determining membrane protein structure, the elastic material properties of even pure lipid nanodiscs (i.e., without embedded proteins) have not yet been quantitatively investigated. A major hurdle is due to the inherently non-local treatment of the elastic properties of lipid systems implemented by most existing methods, both experimental and computational. In addition, these methods are best suited for very large “infinite” size lipidic assemblies, or ones that contain periodicity, in the case of simulations. We have previously described a computational analysis of molecular dynamics simulations designed to overcome these limitations, so that it allows quantification of the bending rigidity (K_C) and tilt moduli (κ_t) on a local scale even for finite, non-periodic systems, such as lipid nanodiscs. Here we use this computational approach to extract values of K_C and κ_t for a set of lipid nanodisc systems that vary in size and lipid composition. We find that the material properties of lipid nanodiscs are different from those of infinite bilayers of corresponding lipid composition, highlighting the effect of nanodisc confinement. Nanodiscs tend to show higher stiffness than their corresponding macroscopic bilayers, and moreover, their material properties vary spatially within them. For small-size MSP1 nanodiscs, the stiffness decreases radially, from a value that is larger in their center than the moduli of the corresponding bilayers by a factor of ~2-3. The larger nanodiscs (MSP1E3D1 and MSP2N2) show milder spatial changes of moduli that are composition dependent and can be maximal in the center or at some distance from it. These trends in moduli correlate with spatially varying structural properties, including the area per lipid and the nanodisc thickness. Finally, as has previously been reported, nanodiscs tend to show deformations from perfectly flat circular geometries to varying degrees, depending on size and lipid composition. The modulations of lipid elastic properties that we find should be carefully considered when making structural and functional inferences concerning embedded proteins.

In calculating transfer free energies of solvated substances, the coexisting crystal state is often taken as the reference. Furthermore, the free energy of this reference state is often assumed to remain constant upon changes made in solution. Yet little is known about the way added cosolutes impact the thermodynamic stability of the out-of-solution crystal phase. To provide insight into the changes in the activity of the coexisting solid state, we used caffeine, a well-studied hydrophobic compound that forms a hydrated crystal in saturated aqueous solutions. By using X-ray powder diffraction, we found that cosolutes, such as trehalose, sucrose, sodium sulfate, and polyethylene glycol (PEG) alter the unit cell volume of crystalline caffeine, in a concentration dependent manner. The dehydration of solid caffeine translates into an overall increase in its free energy, which can be directly calculated as the reversible ΠV work required to compress the crystal. We determined that trehalose, sucrose, and sodium sulfate increase the free energy of the solid, while PEG decreases it. For 2 mol/kg trehalose, this change in free energy corresponds to 17 % of the total change in solvation free energy of monomeric caffeine. Although our results indicate that cosolutes modify the free energy of the solid less than that of the solvated state, this effect is non negligible and measurable, suggesting that it should generally be taken into account as a contribution to changes in solubility, particularly whenever the solid phase is hydrated.

Deep eutectic mixtures are a promising sustainable and diverse class of tunable solvents that hold great promise for various green chemical and technological processes. Many deep eutectic solvents (DES) are hygroscopic and find use in applications with varying extents of hydration, hence urging a profound understanding of changes in the nanostructure of DES with water content. Here, we report on molecular dynamics simulations of the quintessential choline chloride–urea mixture, using a newly parametrized force field with scaled charges to account for physical properties of hydrated DES mixtures. These simulations indicate that water changes the nanostructure of solution even at very low hydration. We present a novel approach that uses convex constrained analysis to dissect radial distribution functions into base components representing different modes of local association. Specifically, DES mixtures can be deconvoluted locally into two dominant competing nanostructures, whose relative prevalence (but not their salient structural features) change with added water over a wide concentration range, from dry up to ∼30 wt % hydration. Water is found to be associated strongly with several DES components but remarkably also forms linear bead-on-string clusters with chloride. At high water content (beyond ∼50 wt % of water), the solution changes into an aqueous electrolyte-like mixture. Finally, the structural evolution of the solution at the nanoscale with extent of hydration is echoed in the DES macroscopic material properties. These changes to structure, in turn, should prove important in the way DES acts as a solvent and to its interactions with additive components.