How a Chain Can Be Extended While Its Bonds Are Compressed.

Extending polymer chains results in a positive chain tension, fch, primarily due to conformational restrictions. At the level of individual bonds, however, tension, fb, is either negative or positive and depends on both chain tension and bulk pressure. Typically, the chain and bond tension are assumed to be directly related. In specific systems, however, this dependence may not be intuitive, whereby fch increases while fb decreases; i.e., the entire chain is extended while bonds are compressed. Specifically, increasing the grafting density of a polymer brush results in chain extension along the direction perpendicular to the grafting surface while the underlying bonds are compressed. Similarly, upon compression of polymer networks, the extension of chains oriented in the "free" direction increases while their bonds are getting more compressed. We demonstrate this phenomenon in molecular dynamics simulations and explain it by the fact that the pressure contribution to fb is dominant over a wide range of network deformations and brush grafting densities.

Facile mechanochemical cycloreversion of polymer cross-linkers enhances tear resistance.

The mechanical properties of covalent polymer networks often arise from the permanent end-linking or cross-linking of polymer strands, and molecular linkers that break more easily would likely produce materials that require less energy to tear. We report that cyclobutane-based mechanophore cross-linkers that break through force-triggered cycloreversion lead to networks that are up to nine times as tough as conventional analogs. The response is attributed to a combination of long, strong primary polymer strands and cross-linker scission forces that are approximately fivefold smaller than control cross-linkers at the same timescales. The enhanced toughness comes without the hysteresis associated with noncovalent cross-linking, and it is observed in two different acrylate elastomers, in fatigue as well as constant displacement rate tension, and in a gel as well as elastomers.

Elasticity of Slide-Ring Gels.

Slide-ring gels are polymer networks with cross-links that can slide along the chains. In contrast to conventional unentangled networks with cross-links fixed along the chains, the slide-ring networks are strain-softening and distribute tension much more uniformly between their strands due to the so-called "pulley effect". The sliding of cross-links also reduces the elastic modulus in comparison with the modulus of conventional networks with the same number density of cross-links and elastic strands. We develop a single-chain model to account for the redistribution of monomers between network strands of a primary chain. This model takes into account both the pulley effect and fluctuations in the number of monomers per network strand. The pulley effect leads to modulus reduction and uniform tension redistribution between network strands, while fluctuations in the number of strand monomers dominate the strain-softening, the magnitude of which decreases upon network swelling and increases upon deswelling.

Macromolecular Crowding Is More than Hard-Core Repulsions.

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.

ß-Hairpin Miniprotein Stabilization in Trehalose Glass Is Facilitated by an Emergent Compact Non-Native State.

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.

Molecular Characterization of Polymer Networks.

Polymer networks are complex systems consisting of molecular components. Whereas the properties of the individual components are typically well understood by most chemists, translating that chemical insight into polymer networks themselves is limited by the statistical and poorly defined nature of network structures. As a result, it is challenging, if not currently impossible, to extrapolate from the molecular behavior of components to the full range of performance and properties of the entire polymer network. Polymer networks therefore present an unrealized, important, and interdisciplinary opportunity to exert molecular-level, chemical control on material macroscopic properties. A barrier to sophisticated molecular approaches to polymer networks is that the techniques for characterizing the molecular structure of networks are often unfamiliar to many scientists. Here, we present a critical overview of the current characterization techniques available to understand the relation between the molecular properties and the resulting performance and behavior of polymer networks, in the absence of added fillers. We highlight the methods available to characterize the chemistry and molecular-level properties of individual polymer strands and junctions, the gelation process by which strands form networks, the structure of the resulting network, and the dynamics and mechanics of the final material. The purpose is not to serve as a detailed manual for conducting these measurements but rather to unify the underlying principles, point out remaining challenges, and provide a concise overview by which chemists can plan characterization strategies that suit their research objectives. Because polymer networks cannot often be sufficiently characterized with a single method, strategic combinations of multiple techniques are typically required for their molecular characterization.

Restructuring a Deep Eutectic Solvent by Water: The Nanostructure of Hydrated Choline Chloride/Urea.

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.

Properties of Aqueous Trehalose Mixtures: Glass Transition and Hydrogen Bonding.

Trehalose is a naturally occurring disaccharide known to remarkably stabilize biomacromolecules in the biologically active state. The stabilizing effect is typically observed over a large concentration range and affects many macromolecules including proteins, lipids, and DNA. Of special interest is the transition from aqueous solution to the dense and highly concentrated glassy state of trehalose that has been implicated in bioadaptation of different organisms toward desiccation stress. Although several mechanisms have been suggested to link the structure of the low water content glass with its action as an exceptional stabilizer, studies are ongoing to resolve which are most pertinent. Specifically, the role that hydrogen bonding plays in the formation of the glass is not well resolved. Here we model aqueous trehalose mixtures over a wide concentration range, using molecular dynamics simulations with two available force fields. Both force fields indicate glass transition temperatures and osmotic pressures that are close to experimental values, particularly at high trehalose contents. We develop and employ a methodology that allows us to analyze the thermodynamics of hydrogen bonds in simulations at different water contents and temperatures. Remarkably, this analysis is able to link the liquid to glass transition with changes in hydrogen bond characteristics. Most notably, the onset of the glassy state can be quantitatively related to the transition from weakly to strongly correlated hydrogen bonds. Our findings should help resolve the properties of the glass and the mechanisms of its formation in the presence of added macromolecules.

A Change in ECM Composition Affects Sensory Organ Mechanics and Function.

Proprioception requires the transduction of muscle-generated deformations into sensory neuronal impulses. In proprioceptive organs, the mechanical coupling between the sensory neuron and the muscle is mediated by a connective structure composed of accessory cells and an extracellular matrix (ECM). Here, we use the fly chordotonal organ (ChO) to investigate how the mechanical properties of the connective element affect mechanosensing. We show that the loss of Pericardin, a major constituent of the ChO ECM, alters the mechanical properties of the ChO resulting in short-wavelength buckling of the accessory cells upon muscle contraction and low compressive strain within the organ. We explain these results using a simplified theoretical model of an elastic beam interacting with an elastic network under a compressive force. We further demonstrate that the transition from compression to bending interferes with the ability of the accessory cells to propagate muscle-generated deformations correctly to the neuron and hence with proper sensing.

Mechanical Communication Acts as a Noise Filter.

Cells can communicate mechanically by responding to mechanical deformations generated by their neighbors. Here, we describe a new role for mechanical communication by demonstrating that mechanical coupling between cells acts as a signaling cue that reduces intrinsic noise in the interacting cells. We measure mechanical interaction between beating cardiac cells cultured on a patterned flexible substrate and find that beat-to-beat variability decays exponentially with coupling strength. To demonstrate that such noise reduction is indeed a direct consequence of mechanical coupling, we reproduce the exponential decay in an assay where a beating cell interacts mechanically with an artificial stochastic 'mechanical cell'. The mechanical cell consists of a probe that mimics the deformations generated by a stochastically beating neighboring cardiac cell. We show that noise reduction through mechanical coupling persists long after stimulation stops and identify microtubule integrity, NOX2, and CaMKII as mediators of noise reduction.

Talking over the extracellular matrix: How do cells communicate mechanically?

Communication between cells enables them to coordinate their activity and is crucial for the differentiation, development, and function of tissues and multicellular organisms. Cell-cell communication is discussed almost exclusively as having a chemical or electrical origin. Only recently, a new mode of cell communication was elucidated: mechanical communication through the extracellular matrix (ECM). Cells can communicate mechanically by responding either to mechanical deformations generated by their neighbors or to a change in the mechanical properties of the ECM induced by a neighboring cell. This newly resolved mode of communication possesses unique features that complement the cellular ability to receive and share information, and to consequently act in a cooperative way with surrounding cells. Herein, we review several examples of mechanical communication, discuss their unique properties, and comment on the major challenges facing the field.

Revisiting Hydrogen Bond Thermodynamics in Molecular Simulations.

In processes involving aqueous solutions and in almost every biomolecular interaction, hydrogen bonds play important roles. Though weak compared to the covalent bond, hydrogen bonds modify the stability and conformation of numerous small and large molecules and modulate their intermolecular interactions. We propose a simple methodology for extracting hydrogen bond strength from atomistic level simulations. The free energy associated with hydrogen bond formation is conveniently calculated as the reversible work required to reshape a completely random pair probability distribution reference state into the one found in simulations where hydrogen bonds are formed. Requiring only the probability density distribution of donor-acceptor pairs in the first solvation shell of an electronegative atom, the method uniquely defines the free energy, entropy, and enthalpy of the hydrogen bond. The method can be easily extended to molecules other than water and to multiple component mixtures. We demonstrate and apply this methodology to hydrogen bonds that form in molecular dynamics simulations between water molecules in pure water, as well as to bonds formed between different molecules in a binary mixture of a sugar (trehalose) and water. Finally, we comment on how the method should be useful in assessing the role of hydrogen bonds in different molecular mechanisms.

Nonclassical Crystal Growth as Explanation for the Riddle of Polarity in Centrosymmetric Glycine Crystals.

The riddle of anomalous polar behavior of the centrosymmetric crystal of α-glycine is resolved by the discovery of a polar, several hundred nanometer thick hydrated layer, created at the {010} faces during crystal growth. This layer was detected by two independent pyroelectric analytical methods: (i) periodic temperature change technique (Chynoweth) at ambient conditions and (ii) contactless X-ray photoelectron spectroscopy under ultrahigh vacuum. The total polarization of the surface layer is extremely large, yielding ≈1 µC·cm-2, and is preserved in ultrahigh vacuum, but disappears upon heating to 100 °C. Molecular dynamics simulations corroborate the formation of polar hydrated layers at the sub-microsecond time scale, however with a thickness of only several nanometers, not several hundred. This inconsistency might be reconciled by invoking a three-step nonclassical crystal growth mechanism comprising (i) docking of clusters from the supersaturated solution onto the evolving crystal, (ii) surface recognition and polar induction, and (iii) annealing and dehydration, followed by site-selective recrystallization.

Properties of Polyvinylpyrrolidone in a Deep Eutectic Solvent.

Deep eutectic solvents (DES) are mixtures of two or more components with high melting temperatures, which form a liquid at room temperature. These DES hold great promise as green solvents for chemical processes, as they are inexpensive and environmentally friendly. Specifically, they present a unique solvating environment to polymers that is different from water. Here, we use small angle neutron scattering to study the polymer properties of the common, water-soluble, polyvinylpyrrolidone (PVP) in the prominent DES formed by a 1:2 molar mixture of choline chloride and urea. We find that the polymer adopts a slightly different structure in DES than in water, so that at higher concentrations the polymer favors a more expanded conformation compared to the same concentration in water. Yet, the osmotic pressure of PVP solutions in DES is very similar to that in water, indicating that both solvents are of comparable quality and that the DES components interact favorably with PVP. The osmotic pressure measurements within this novel class of promising solvents should be of value toward future technological applications as well as for osmotic stress experiments in nonaqueous environments.

Osmolyte Induced Changes in Peptide Conformational Ensemble Correlate with Slower Amyloid Aggregation: A Coarse-Grained Simulation Study.

Stabilizing osmolytes are known to impact the process of amyloid aggregation, often altering aggregation kinetics. Recent evidence further suggests that osmolytes modify the peptide conformational dynamics, as well as change the physical characteristics of assembling amyloid fibrils. To resolve how these variations emerge on the molecular level, we simulated the initial aggregation steps of an amyloid-forming peptide in the presence and absence of the osmolyte sorbitol, a naturally occurring polyol. To this end, a coarse-grained force field was extended and implemented to access larger aggregate sizes and longer time scales. The force field optimization procedure placed emphasis on calibrating the solution thermodynamics of sorbitol, the aggregating peptide in its monomeric form, and the interaction of both of these components with each other and with water. Our simulations show a difference in aggregation kinetics and structural parameters in the presence of sorbitol compared to water, which qualitatively agree well with our experimentally resolved aggregation kinetics of the same peptide. The kinetic changes induced by sorbitol can be traced in our simulations to changes in monomer conformations resulting from osmolyte presence. These translate into changes in peptide conformations within the aggregated clusters and into differences in rates of monomer nucleation and of association to formed fibrils. We find that, compared to pure water as solvent, the presence of sorbitol induces formation of more aggregates each containing fewer peptides, with an increased tendency toward parallel interpeptide contacts.

Macromolecular Stabilization by Excluded Cosolutes: Mean Field Theory of Crowded Solutions.

We propose a mean field theory to account for the experimentally determined temperature dependence of protein stabilization that emerges in solutions crowded by preferentially excluded cosolutes. Based on regular solution theory and employing the Flory-Huggins approximation, our model describes cosolutes in terms of their size, and two temperature-dependent microscopic parameters that correspond to macromolecule-cosolute and bulk solution interactions. The theory not only predicts a "depletion force" that can account for the experimentally observed stabilization of protein folding or association in the presence of excluded cosolutes but also predicts the full range of associated entropic and enthalpic components. Remarkably, depending on cosolute identity and in accordance with experiments, the theory describes entropically as well as enthalpically dominated depletion forces, even those disfavored by entropy. This emerging depletion attraction cannot be simply linked to molecular volumes. Instead, the relevant parameter is an effective volume that represents an interplay between solvent, cosolute, and macromolecular interactions. We demonstrate that the apparent depletion free energy is often accompanied by significant yet compensating entropy and enthalpy terms that, although having a net zero contribution to stabilization, can obscure the underlying molecular mechanism. This study underscores the importance of including often-neglected free energy terms that correspond to solvent-cosolute and cosolute-macromolecule interactions, which for most typical cosolutes are expected to be temperature dependent. We propose that experiments specifically aimed at resolving the temperature-dependence of cosolute exclusion from macromolecular surfaces should help reveal the full range of the underlying molecular mechanisms of the depletion force.

Origin of Enthalpic Depletion Forces.

Solutes excluded from macromolecules or colloids are known to drive depletion attractions. The established Asakura-Oosawa model, as well as subsequent theories aimed at explaining the effects of macromolecular crowding, attribute depletion forces to diminished hard-core excluded volume upon compaction, and hence predict depletion forces dominated by entropy. However, recent experiments measuring the effect of preferentially excluded solutes on protein folding and macromolecular association find these forces can also be enthalpic. We use simulations of macromolecular association in explicit binary cosolute-solvent mixtures, with solvent and cosolute intermolecular interactions that go beyond hard-cores, to show that not all cosolutes conform to the established entropically dominated model. We further demonstrate how the enthalpically dominated depletion forces that we find can be well described within an Asakura-Oosawa like model provided that the hard-core macromolecule-cosolute potential of mean force is augmented by a "soft" step-like repulsion.

Diversity in the mechanisms of cosolute action on biomolecular processes.

Numerous cellular cosolutes significantly impact the way that proteins and other biomacromolecules act and interact. We have followed the thermodynamic effect of several cosolute classes, including polymers, cellular osmolytes, and inorganic salts, on the stability of biomolecular folding and complexation. By comparing changes in free energy, enthalpy, and entropy upon cosolutes addition for these processes, we identify several thermodynamically distinct mechanisms. Surprisingly, even while many cosolutes display similar scaling of the change in stabilizing free energy with their concentration, a breakdown of this free energy into enthalpic and entropic contributions distinguishes different families of cosolutes. We discuss how these "thermodynamic fingerprints" can direct towards possible underlying mechanisms that govern the cosolute effect.

Linking trehalose self-association with binary aqueous solution equation of state.

In aqueous solutions, trehalose possesses a high propensity to form hydrogen bonds with water as well as other trehalose molecules. This hydrogen bonding not only affects water structure but also promotes extensive concentration dependent aggregation of trehalose molecules, which may impact trehalose's role as a protective cosolute to biomacromolecules. To study the association of trehalose in aqueous solutions over a wide concentration range, we used molecular dynamics simulations based on two different force fields, as well as vapor pressure osmometry. By analyzing trehalose cluster size and fractal dimension in simulations, we estimate the cluster percolation threshold at 1.5-2.2 m. Experimentally, trehalose solutions showed positive deviations from ideal van't Hoff's law that grew with concentration. These variations in osmotic pressure can be explained using a simple equation of state that accounts for the repulsive excluded volume interactions between trehalose molecules as well as attractions reflected in sugar clustering. We find that simulations with both applied force fields result in reasonable representations of the solution equation of state. However, in contrast to experiments, the balance between the repulsive and attractive trehalose-trehalose interactions in simulations results in a slightly negative deviation from ideality, probably due to the moderately overaggregative nature of the force fields used.

The impact of polyols on water structure in solution: a computational study.

Using molecular dynamics simulations, we study the effect of polyalcohols on water structuring in concentrated solutions, comparing six different polyols that vary in the number of hydroxyl groups and internal structure. For all polyols, we find that the hydrogen bond network order, as assessed by changes in the tetrahedral order parameter, is distorted in the binary solutions as compared with that of pure water and depends on the number of hydroxyl groups and the polyol conformation. While the total number of hydrogen bonds is only slightly reduced relative to that found in pure water, we find that hydrogen bonds that form with polyols tend to be less linear than hydrogen bonds formed between water molecules. We suggest that this reflects the competition between water and polyol molecules for hydrogen bonding with surrounding waters and offer a link between this competition and the resulting disorder that follows polyol solvation. The conclusions of this study should help shed light on the action that polyols can have as stabilizers to other macromolecules such as proteins in solution.