Triblock Proteins with Weakly Dimerizing Terminal Blocks and an Intrinsically Disordered Region for Rational Design of Condensate Properties.

Condensates are molecular assemblies that are formed through liquid-liquid phase separation and play important roles in many biological processes. The rational design of condensate formation and their properties is central to applications, such as biosynthetic materials, synthetic biology, and for understanding cell biology. Protein engineering is used to make a triblock structure with varying terminal blocks of folded proteins on both sides of an intrinsically disordered mid-region. Dissociation constants are determined in the range of micromolar to millimolar for a set of proteins suitable for use as terminal blocks. Varying the weak dimerization of terminal blocks leads to an adjustable tendency for condensate formation while keeping the intrinsically disordered region constant. The dissociation constants of the terminal domains correlate directly with the tendency to undergo liquid-liquid phase separation. Differences in physical properties, such as diffusion rate are not directly correlated with the strength of dimerization but can be understood from the properties and interplay of the constituent blocks. The work demonstrates the importance of weak interactions in condensate formation and shows a principle for protein design that will help in fabricating functional condensates in a predictable and rational way.

Ion-Specific Effects on Ion and Polyelectrolyte Solvation.

Ion-specific effects on aqueous solvation of monovalent counter ions, Na + ${^+ }$ , K + ${^+ }$ , Cl - ${^- }$ , and Br - ${^- }$ , and two model polyelectrolytes (PEs), poly(styrene sulfonate) (PSS) and poly(diallyldimethylammonium) (PDADMA) were here studied with ab initio molecular dynamics (AIMD) and classical molecular dynamics (MD) simulations based on the OPLS-aa force-field which is an empirical fixed point-charge force-field. Ion-specific binding to the PE charge groups was also characterized. Both computational methods predict similar response for the solvation of the PEs but differ notably in description of ion solvation. Notably, AIMD captures the experimentally observed differences in Cl - ${^- }$ and Br - ${^- }$ anion solvation and binding with the PEs, while the classical MD simulations fail to differentiate the ion species response. Furthermore, the findings show that combining AIMD with the computationally less costly classical MD simulations allows benefiting from both the increased accuracy and statistics reach.

Interactions between Rigid Polyelectrolytes Mediated by Ordering and Orientation of Multivalent Nonspherical Ions in Salt Solutions.

Multivalent ions in solutions with polyelectrolytes (PEs) induce electrostatic correlations that can drastically change ion distributions around the PEs and their mutual interactions. Using coarse-grained molecular dynamics simulations, we show how in addition to valency, ion shape and concentration can be harnessed as tools to control rigid like-charged PE-PE interactions. We demonstrate a correlation between the orientational ordering of aspherical ions and how they mediate the effective PE-PE attraction induced by multivalency. The interaction type, strength, and range can thus be externally controlled in ionic solutions. Our results can be used as generic guidelines to tune the self-assembly of like-charged polyelectrolytes by variation of the characteristics of the ions.

A Perspective on the Glass Transition and the Dynamics of Polyelectrolyte Multilayers and Complexes.

Polyelectrolyte multilayers (PEMs) or polyelectrolyte complexes (PECs), formed by layer-by-layer assembly or the mixing of oppositely charged polyelectrolytes (PEs) in aqueous solution, respectively, have potential applications in health, energy, and the environment. PEMs and PECs are very tunable because their structure and properties are influenced by factors such as pH, ionic strength, salt type, humidity, and temperature. Therefore, it is increasingly important to understand how these factors affect PECs and PEMs on a molecular level. In this Feature Article, we summarize our contributions to the field in the development of approaches to quantify the swelling, thermal properties, and dynamic mechanical properties of PEMs and PECs. First, the role of water as a plasticizer and in the glass-transition temperature (Tg) in both strong poly(diallyldimethylammonium)/poly(sodium 4-styrenesulfonate) (PDADMA/PSS) and weak poly(allylamine hydrochloride)/poly(acrylic acid) (PAH/PAA) systems is presented. Then, factors influencing the dynamics of PECs and PEMs are discussed. We also reflect on the swelling of PEMs in response to different salts and solvent additives. Last, the nature of water's microenvironment in PEMs/PECs is discussed. A special emphasis is placed on experimental techniques, along with molecular simulations. Taken together, this review presents an outlook and offers recommendations for future research directions, such as studying the additional effects of hydrogen-bonding hydrophobic interactions.

Spidroins under the Influence of Alcohol: Effect of Ethanol on Secondary Structure and Molecular Level Solvation of Silk-Like Proteins.

Future sustainable materials based on designer biomolecules require control of the solution assembly, but also interfacial interactions. Alcohol treatments of protein materials are an accessible means to this, making understanding of the process at the molecular level of seminal importance. We focus here on the influence of ethanol on spidroins, the main proteins of silk. By large-scale atomistically detailed molecular dynamics (MD) simulations and interconnected experiments, we characterize the protein aggregation, secondary structure changes, molecular level origins of them, and solvation environment changes for the proteins, as induced by ethanol as a solvation additive. The MD and circular dichoroism (CD) findings jointly show that ethanol promotes ordered structure in the protein molecules, leading to an increase of helix content and turns but also increased aggregation, as revealed by dynamic light scattering (DLS) and light microscopy. The structural changes correlate at the molecular level with increased intramolecular hydrogen bonding. The simulations reveal that polar amino acids, such as glutamine and serine, are most influenced by ethanol, whereas glycine residues are most prone to be involved in the ethanol-induced secondary structure changes. Furthermore, ethanol engages in interactions with the hydrophobic alanine-rich regions of the spidroin, significantly decreasing the hydrophobic interactions of the protein with itself and its surroundings. The protein solutes also change the microstructure of water/ethanol mixtures, essentially decreasing the level of larger local clustering. Overall, the work presents a systematic characterization of ethanol effects on a widely used, common protein type, spidroins, and generalizes the findings to other intrinsically disordered proteins by pinpointing the general features of the response. The results can aid in designing effective alcohol treatments for proteins, but also enable design and tuning of protein material properties by a relatively controllable solvation handle, the addition of ethanol.

From microemulsion phase diagrams to hydrophilicity and hydration controlled adsorption: a dissipative particle dynamics modelling study of phospholipid assembly in bio oils.

We explore here the assembly and adsorption response of a ternary bio oil-phospholipid-water system via dissipative particle dynamics (DPD) simulations. The mesoscale, particle-based modelling approach allows the examination of large-scale self-assembly response of dipalmitoylphosphatidylcholine (DPPC) phospholipids in a model bio oil solvent (modelled by triglycerides) in the presence of varying amounts of water. We report the reverse micellar and microemulsion assembly phase diagrams of the ternary mixture, verifying the model against literature data. The results show water content vs phospholipid concentration dependent transitions in reverse micellar to network-like and various lamellar phases in bulk assembly. Examination of the DPPC adsorption to smooth, homogeneous adsorbate surfaces of differing polarity reveals that phospholipid adsorption response transitions between discrete assemblies on polyethylene-like hydrophobic to continuous coating on mica-like hydrophilic substrates as the function of phospholipid and water concentrations. The significance of the work is that the presented model for phospholipid assembly in apolar solvents is capable of predicting accurately large scale assembly response and morphology changes including adsorption response in terms of system variables. The presented parametrization and verification information of the model enable readily extending the approach to other systems. The work provides computational access for tuning lipid-based microemulsion systems and their adsorption.

Dioctyl sodium sulfosuccinate surfactant self-assembly dependency of solvent hydrophilicity: a modelling study.

The self-assembly of dioctyl sodium sulfosuccinate (AOT) model surfactant in solvent environments of differing polarity is examined by means of dissipative particle dynamics (DPD) bead model parametrized against Hildebrand solubility parameters from atomistic molecular dynamics (MD) simulations. The model predicts that in hydrophobic solvents (e.g. dodecane) the surfactant forms small (Nagg ∼ 8) reverse micellar aggregates, while in a solvent corresponding to water lamellar assembly takes place, in good agreement with literature structural parameters. Interestingly, solvents of intermediate polarity lead to formation of large, internally structured aggregates. In these, the surfactant headgroups cluster within the aggregate, surrounded by a continuous phase formed by the hydrocarbon tails. We show that the partitioning of the headgroups between the aggregate surface layer and the inner clustered phase depends primarily on solvent polarity, and can be controlled by the solvent, but also system composition. Finally, we compare the DPD assembly response to simplified effective interaction potentials derived at dilute concentration limit for the interactions. The comparison reveals that the simplified effective potential descriptions provide good level of insight on the assembly morphologies, despite drastic, isotropic interactions simplification involved.

pH dependence of the assembly mechanism and properties of poly(L-lysine) and poly(L-glutamic acid) complexes.

We show by extensive experimental characterization combined with molecular simulations that pH has a major impact on the assembly mechanism and properties of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) complexes. A combination of dynamic light scattering (DLS) and laser Doppler velocimetry (LDV) is used to assess the complexation, charge state, and other physical characteristics of the complexes, isothermal titration calorimetry (ITC) is used to examine the complexation thermodynamics, and circular dichroism (CD) is used to extract the polypeptides' secondary structure. For enhanced analysis and interpretation of the data, analytical ultracentrifugation (AUC) is used to define the precise molecular weights and solution association of the peptides. Molecular dynamics simulations reveal the associated intra- and intermolecular binding changes in terms of intrinsic vs. extrinsic charge compensation, the role of hydrogen bonding, and secondary structure changes, aiding in the interpretation of the experimental data. We combine the data to reveal the pH dependency of PLL/PGA complexation and the associated molecular level mechanisms. This work shows that not only pH provides a means to control complex formation but also that the associated changes in the secondary structure and binding conformation can be systematically used to control materials assembly. This gives access to rational design of peptide materials via pH control.

Liquid-Liquid Phase Separation and Assembly of Silk-like Proteins is Dependent on the Polymer Length.

Phase transitions have an essential role in the assembly of nature's protein-based materials into hierarchically organized structures, yet many of the underlying mechanisms and interactions remain to be resolved. A central question for designing proteins for materials is how the protein architecture and sequence affects the nature of the phase transitions and resulting assembly. In this work, we produced 82 kDa (1×), 143 kDa (2×), and 204 kDa (3×) silk-mimicking proteins by taking advantage of protein ligation by SpyCatcher/Tag protein-peptide pair. We show that the three silk proteins all undergo a phase transition from homogeneous solution to assembly formation. In the assembly phase, a length- and concentration-dependent transition between two distinct assembly morphologies, one forming aggregates and another coacervates, exists. The coacervates showed properties that were dependent on the protein size. Computational modeling of the proteins by a bead-spring model supports the experimental results and provides us a possible mechanistic origin for the assembly transitions based on architectures and interactions.

Interaction between two polyelectrolytes in monovalent aqueous salt solutions.

We use the recently developed soft-potential-enhanced Poisson-Boltzmann (SPB) theory to study the interaction between two parallel polyelectrolytes (PEs) in monovalent ionic solutions in the weak-coupling regime. The SPB theory is fitted to ion distributions from coarse-grained molecular dynamics (MD) simulations and benchmarked against all-atom MD modelling for poly(diallyldimethylammonium) (PDADMA). We show that the SPB theory is able to accurately capture the interactions between two PEs at distances beyond the PE radius. For PDADMA positional correlations between the charged groups lead to locally asymmetric PE charge and ion distributions. This gives rise to small deviations from the SPB prediction that appear as short-range oscillations in the potential of mean force. Our results suggest that the SPB theory can be an efficient way to model interactions in chemically specific complex PE systems.

Modified Poisson-Boltzmann theory for polyelectrolytes in monovalent salt solutions with finite-size ions.

We present a soft-potential-enhanced Poisson-Boltzmann (SPB) theory to efficiently capture ion distributions and electrostatic potential around rodlike charged macromolecules. The SPB model is calibrated with a coarse-grained particle-based model for polyelectrolytes (PEs) in monovalent salt solutions as well as compared to a full atomistic molecular dynamics simulation with the explicit solvent. We demonstrate that our modification enables the SPB theory to accurately predict monovalent ion distributions around a rodlike PE in a wide range of ion and charge distribution conditions in the weak-coupling regime. These include excess salt concentrations up to 1M and ion sizes ranging from small ions, such as Na+ or Cl-, to softer and larger ions with a size comparable to the PE diameter. The work provides a simple way to implement an enhancement that effectively captures the influence of ion size and species into the PB theory in the context of PEs in aqueous salt solutions.

Self-Assembly of Silk-like Protein into Nanoscale Bicontinuous Networks under Phase-Separation Conditions.

Liquid-liquid phase separation of biomacromolecules is crucial in various inter- and extracellular biological functions. This includes formation of condensates to control, e.g., biochemical reactions and structural assembly. The same phenomenon is also found to be critically important in protein-based high-performance biological materials. Here, we use a well-characterized model triblock protein system to demonstrate the molecular level formation mechanism and structure of its condensate. Large-scale molecular modeling supported by analytical ultracentrifuge characterization combined with our earlier high magnification precision cryo-SEM microscopy imaging leads to deducing that the condensate has a bicontinuous network structure. The bicontinuous network rises from the proteins having a combination of sites with stronger mutual attraction and multiple weakly attractive regions connected by flexible, multiconfigurational linker regions. These attractive sites and regions behave as stickers of varying adhesion strength. For the examined model triblock protein construct, the ß-sheet-rich end units are the stronger stickers, while additional weaker stickers, contributing to the condensation affinity, rise from spring-like connections in the flexible middle region of the protein. The combination of stronger and weaker sticker-like connections and the flexible regions between the stickers result in a versatile, liquid-like, self-healing structure. This structure also explains the high flexibility, easy deformability, and diffusion of the proteins, decreasing only 10-100 times in the bicontinuous network formed in the condensate phase in comparison to dilute protein solution. The here demonstrated structure and condensation mechanism of a model triblock protein construct via a combination of the stronger binding regions and the weaker, flexible sacrificial-bond-like network as well as its generalizability via polymer sticker models provide means to not only understand intracellular organization, regulation, and cellular function but also to identify direct control factors for and to enable engineering improved protein and polymer constructs to enhance control of advanced fiber materials, smart liquid biointerfaces, or self-healing matrices for pharmaceutics or bioengineering materials.

Physisorption of bio oil nitrogen compounds onto montmorillonite.

We assess computationally the adsorption of a series of nitrogen containing heterocycles and fatty acid amides from bio-oil on a model clay surface, Na-montmorillonite. The adsorption energies and conformations predicted by atomistic detail molecular dynamics (MD) simulations are compared against density functional theory (DFT) based molecular electrostatic potentials (MEP) and Hirshfeld, AIM, Merz-Singh-Kollman, and ChelpG charges. MD predicts systematically adsorption via cation bridging with adsorption strength of the heterocycles following purine > pyridine > imidazole > pyrrole > indole > quinoline. The fatty acid amides adsorption strength follows the steric availability and bulkiness of the head group. A comparison against the DFT calculations shows that MEP predicts adsorption geometries and the MD simulations reproduce the conformations for single adsorption site species. However, the DFT derived charge distibutions show that MD force-fields with non-polarizable fixed partial charge representations parametrized for aqueous environments cannot be used in apolar solvent environments without careful accuracy considerations. The overall trends in adsorption energies are reproduced by the Charmm GenFF employed in the MD simulations but the adsorption energies are systematically overestimated in this apolar solvent environment. The work has significance both for revealing nitrogen compound adsorption trends in technologically relevant bio oil environments but also as a methodological assessment revealing the limits of state of the art biomolecular force-fields and simulation protocols in apolar bioenvironments.

Self-assembly of binary solutions to complex structures.

Self-assembly in natural and synthetic molecular systems can create complex aggregates or materials whose properties and functionalities rise from their internal structure and molecular arrangement. The key microscopic features that control such assemblies remain poorly understood, nevertheless. Using classical density functional theory, we demonstrate how the intrinsic length scales and their interplay in terms of interspecies molecular interactions can be used to tune soft matter self-assembly. We apply our strategy to two different soft binary mixtures to create guidelines for tuning intermolecular interactions that lead to transitions from a fully miscible, liquid-like uniform state to formation of simple and core-shell aggregates and mixed aggregate structures. Furthermore, we demonstrate how the interspecies interactions and system composition can be used to control concentration gradients of component species within these assemblies. The insight generated by this work contributes toward understanding and controlling soft multi-component self-assembly systems. Additionally, our results aid in understanding complex biological assemblies and their function and provide tools to engineer molecular interactions in order to control polymeric and protein-based materials, pharmaceutical formulations, and nanoparticle assemblies.

Fourier transform infrared spectroscopy investigation of water microenvironments in polyelectrolyte multilayers at varying temperatures.

Polyelectrolyte multilayers (PEMs) are thin films formed by the alternating deposition of oppositely charged polyelectrolytes. Water plays an important role in influencing the physical properties of PEMs, as it can act both as a plasticizer and swelling agent. However, the way in which water molecules distribute around and hydrate ion pairs has not been fully quantified with respect to both temperature and ionic strength. Here, we examine the effects of temperature and ionic strength on the hydration microenvironments of fully immersed poly(diallyldimethylammonium)/polystyrene sulfonate (PDADMA/PSS) PEMs. This is accomplished by tracking the OD stretch peak using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy at 0.25-1.5 M NaCl and 35-70 °C. The OD stretch peak is deconvoluted into three peaks: (1) high frequency water, which represents a tightly bound microenvironment, (2) low frequency water, which represents a loosely bound microenvironment, and (3) bulk water. In general, the majority of water absorbed into the PEM exists in a bound state, with little-to-no bulk water observed. Increasing temperature slightly reduces the amount of absorbed water, while addition of salt increases the amount of absorbed water. Finally, a van't Hoff analysis is applied to estimate the enthalpy (11-22 kJ mol-1) and entropy (48-79 kJ mol-1 K-1) of water exchanging from low to high frequency states.

Shape and Phase Transitions in a PEGylated Phospholipid System.

Poly(ethylene glycol) (PEG) polymers and PEG-conjugated lipids are widely used in bioengineering and drug transport applications. A PEG layer in a drug carrier increases hydrophilic repulsion, inhibits membrane fusion and serum opsonin interactions, and prolongs the storage and circulation time. It can also change the carrier shape and have an influence on many properties related to the content release of the carrier. In this paper, we focus on the physicochemical effects of PEGylation in the lipid bilayer. We introduce laurdanC as a fluorophore for shape recognition and phase transition detection. Together with laurdanC, cryogenic transmission electron microscopy, differential scanning calorimetry, molecular dynamics simulations, and small-angle X-ray scattering/wide-angle X-ray scattering, we acquire information of the particle/bilayer morphology and phase behavior in systems containing 1,2-dipalmitoyl- sn-glycero-3-phosphocholine:1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG(2000) with different fractions. We find that PEGylation leads to two important and potentially usable features of the system. (1) Spherical vesicles present a window of elevated chain-melting temperatures and (2) lipid packing shape-controlled liposome-to-bicelle transition. The first finding is significant for targets requiring multiple release sequences and the second enables tuning the release by composition and the PEG polymer length. Besides drug delivery systems, the findings can be used in other smart soft materials with trigger-polymers as well.

Phospholipid-Based Reverse Micelle Structures in Vegetable Oil Modified by Water Content, Free Fatty Acid, and Temperature.

Colloidal assemblies of phospholipids in oil are known to be highly sensitive to changes in system composition and temperature. Despite the fundamental biological and high industrial relevance of these aggregates, the mechanisms behind the structural changes, especially in real oils, are not well understood. In this work, small-angle X-ray scattering (SAXS) was combined with molecular dynamics simulations to characterize the effects of oleic acid, water, and temperature on self-assembled structures formed by lecithin in rapeseed oil. SAXS showed that adding water to the mixtures caused the precipitation of liquid-crystalline phases with lamellar or hexagonal geometry. The combination of SAXS and molecular dynamics simulations revealed that stable spherical reverse micelles in oil had a core radius of about 2 nm and consisted of approximately 60 phospholipids centered around a core containing water and sugars. The presence of oleic acid improved the stability of reverse micelles against precipitation due to the increase in the water concentration in oil by allowing the reverse micelle cores to expand and accommodate more water. The shape and size of the reverse micelles changed at high temperatures, and irreversible elongation was observed, especially in the presence of oleic acid. The findings show the interdependency of the structure of the reverse micellar aggregates on system composition, in particular, oleic acid and water, as well as temperature. The revealed characteristics of the self-assembled structures have significance in understanding and tuning the properties of vegetable oil-based emulsions, food products, oil purification, and drug delivery systems.

Comparing water-mediated hydrogen-bonding in different polyelectrolyte complexes.

All-atom molecular dynamics simulations are used to investigate the polyelectrolyte-specific influence of hydration and temperature on water diffusion in hydrated polyelectrolyte complexes (PECs). Two model PECs were compared: poly(allylamine hydrochloride) (PAH)-poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium) (PDADMA)-poly(acrylic acid) (PAA). The findings show that the strength of the hydrogen bonding i.e. polyelectrolyte water interaction has enormous influence on the water mobility, which has implications for PEC structure and properties. A 10-fold difference in the average water diffusion coefficient between PAH-PSS and PDADMA-PAA PECs at the same hydration level is observed. The vast majority of the water molecules hydrating the PDADMA-PAA PECs, for hydrations in the range of 26-38 wt%, are effectively immobilized, whereas for PAH-PSS PECs the amount of immobilized water decreases with hydration. This points to the polyelectrolyte-specific character of the PE-water hydrogen bonding relationship with temperature. PAA-water hydrogen bonds are found to be significantly less sensitive to temperature than for PSS-water. The polyelectrolyte-water interactions, investigated via radial distribution function, hydrogen bond distance and angle distributions, are connected with resulting structure of the PECs. The PDADMA-PAA and PAH-PSS PECs are prepared experimentally and the states of water at different hydration levels is determined using differential scanning calorimetry (DSC). Experiments confirm the differences between PDADMA-PAA and PAH-PSS PECs observed in the theoretical modelling. The results suggest that the initial predictions of the PEC's bonding with water can be based on simple molecular-level considerations.

Surfactant Interactions and Organization at the Gas-Water Interface (CTAB with Added Salt).

We have studied adsorbed layers of cetyltrimethylammonium bromide (CTAB) at air-water interfaces in the presence of added electrolyte. Fast bubble compression/expansion measurements were used to obtain the surface equation of state, i.e., the surface tension vs CTAB surface concentration dependence. We show that while a simple model where the surfactant molecules are assumed to be noninteracting is insufficient to describe the measured response of the surfactant layer, a modified Frumkin equation where the local interactions between the molecular components depend on their surface concentration captures the response. The variation of the effective interactions in the surfactant layer in the model shows that the interactions in the surfactant layer change from effectively repulsive to attractive with increasing surface concentration. Molecular dynamics simulations are performed to probe the origins of the change in the interactions. The simulations indicate that already at low surface concentrations the surfactants aggregate as highly dynamic rafts with surfactant orientation parallel to the interface. Increasing the concentration leads to a change in the assembly morphology at the interface: the surfactant layer thickens and the surfactants sample a range of tilted orientations with respect to the interfacial plane. The change from transient raftlike assemblies to dynamical aggregates at the interface involves a clear increase in the degree of counterion binding: we speculate that the flip of the effective interaction parameter in the model used to interpret the experimental results could result from this. The work here presents basic steps toward a proper understanding of the molecular organization and interactions of surfactants at an air-water interface. This is crucially important in understanding macroscopic properties of surfactant-stabilized systems such as foams, emulsions, and colloidal dispersions.

Particulate Coatings via Evaporation-Induced Self-Assembly of Polydisperse Colloidal Lignin on Solid Interfaces.

Polydisperse smooth and spherical biocolloidal particles were suspended in aqueous media and allowed to consolidate via evaporation-induced self-assembly. The stratification of the particles at the solid-air interface was markedly influenced, but not monotonically, by the drying rate. Cross-sectional imaging via electron microscopy indicated a structured coating morphology that was distinctive from that obtained by using particles with a mono- or bimodal distribution. Segregation patterns were found to derive from the interplay of particle diffusion, interparticle forces, and settling dynamics. Supporting our experimental findings, computer simulations showed an optimal drying rate for achieving maximum segregation. Overall, stratified coatings comprising nano- and microparticles derived from lignin are expected to open opportunities for multifunctional structures that can be designed and predicted on the basis of experimental Péclet numbers and computational order.