Universality in the Structure and Dynamics of Water under Lipidic Mesophase Soft Nanoconfinement.

Water under soft nanoconfinement features physical and chemical properties fundamentally different from bulk water; yet, the multitude and specificity of confining systems and geometries mask any of its potentially universal traits. Here, we advance in this quest by resorting to lipidic mesophases as an ideal nanoconfinement system, allowing inspecting the behavior of water under systematic changes in the topological and geometrical properties of the confining medium, without altering the chemical nature of the interfaces. By combining Terahertz absorption spectroscopy experiments and molecular dynamics simulations, we unveil the presence of universal laws governing the physics of nanoconfined water, recapitulating the data collected at varying levels of hydration and nanoconfinement topologies. This geometry-independent universality is evidenced by the existence of master curves characterizing both the structure and dynamics of simulated water as a function of the distance from the lipid-water interface. Based on our theoretical findings, we predict a parameter-free law describing the amount of interfacial water against the structural dimension of the system (i.e., the lattice parameter), which captures both the experimental and numerical results within the same curve, without any fitting. Our results offer insight into the fundamental physics of water under soft nanoconfinement and provide a practical tool for accurately estimating the amount of nonbulk water based on structural experimental data.

Hierarchical Protofilament Intertwining Rules the Formation of Mixed-Curvature Amyloid Polymorphs.

Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non-zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed-curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed-curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.

Systematic Comparison of Atomistic Force Fields for the Mechanical Properties of Double-Stranded DNA.

The response of double-stranded DNA to external mechanical stress plays a central role in its interactions with the protein machinery in the cell. Modern atomistic force fields have been shown to provide highly accurate predictions for the fine structural features of the duplex. In contrast, and despite their pivotal function, less attention has been devoted to the accuracy of the prediction of the elastic parameters. Several reports have addressed the flexibility of double-stranded DNA via all-atom molecular dynamics, yet the collected information is insufficient to have a clear understanding of the relative performance of the various force fields. In this work, we fill this gap by performing a systematic study in which several systems, characterized by different sequence contexts, are simulated with the most popular force fields within the AMBER family, bcs1 and OL15, as well as with CHARMM36. Analysis of our results, together with their comparison with previous work focused on bsc0, allows us to unveil the differences in the predicted rigidity between the newest force fields and suggests a roadmap to test their performance against experiments. In the case of the stretch modulus, we reconcile these differences, showing that a single mapping between sequence-dependent conformation and elasticity via the crookedness parameter captures simultaneously the results of all force fields, supporting the key role of crookedness in the mechanical response of double-stranded DNA.

Unusual phosphatidylcholine lipid phase behavior in the ionic liquid ethylammonium nitrate.

HYPOTHESIS: The forces that govern lipid self-assembly ionic liquids are similar to water, but their different balance can result in unexpected behaviour. EXPERIMENTS: The self-assembly behaviour and phase equilibria of two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the most common protic ionic liquid, ethylammonium nitrate (EAN) have been investigated as function of composition and temperature by small- and wide-angle X-ray scattering (SAXS/WAXS) and small-angle neutron scattering (SANS). FINDINGS: Both lipids form unusual self-assembly structures and show complex and unexpected phase behaviour unlike that seen in water; DSPC undergoes a gel Lß to crystalline Lc phase transition on warming, while POPC forms worm-like micelles L1 upon dilution. This surprising phase behaviour is attributed to the large size of the EAN ions that solvate the lipid headgroup compared to water changing amphiphile packing. Weaker H-bonding between EAN and lipid headgroups also contributes. These results provide new insight for the design of lipid based nanostructured materials in ionic liquids with atypical properties.

Force-dependent elasticity of nucleic acids.

The functioning of double-stranded (ds) nucleic acids (NAs) in cellular processes is strongly mediated by their elastic response. These processes involve proteins that interact with dsDNA or dsRNA and distort their structures. The perturbation of the elasticity of NAs arising from these deformations is not properly considered by most theoretical frameworks. In this work, we introduce a novel method to assess the impact of mechanical stress on the elastic response of dsDNA and dsRNA through the analysis of the fluctuations of the double helix. Application of this approach to atomistic simulations reveals qualitative differences in the force dependence of the mechanical properties of dsDNA with respect to those of dsRNA, which we relate to structural features of these molecules by means of physically-sound minimalistic models.

Interplay of Hydropathy and Heterogeneous Diffusion in the Molecular Transport within Lamellar Lipid Mesophases.

Lipid mesophases are being intensively studied as potential candidates for drug-delivery purposes. Extensive experimental characterization has unveiled a wide palette of release features depending on the nature of the host lipids and of the guest molecule, as well as on the environmental conditions. However, only a few simulation works have addressed the matter, which hampers a solid rationalization of the richness of outcomes observed in experiments. Particularly, to date, there are no theoretical works addressing the impact of hydropathy on the transport of a molecule within lipid mesophases, despite the significant fraction of hydrophobic molecules among currently-available drugs. Similarly, the high heterogeneity of water mobility in the nanoscopic channels within lipid mesophases has also been neglected. To fill this gap, we introduce here a minimal model to account for these features in a lamellar geometry, and systematically study the role played by hydropathy and water-mobility heterogeneity by Brownian-dynamics simulations. We unveil a fine interplay between the presence of free-energy barriers, the affinity of the drug for the lipids, and the reduced mobility of water in determining the net molecular transport. More in general, our work is an instance of how multiscale simulations can be fruitfully employed to assist experiments in release systems based on lipid mesophases.

A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70.

Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins.

Accurate Sequence-Dependent Coarse-Grained Model for Conformational and Elastic Properties of Double-Stranded DNA.

We introduce MADna, a sequence-dependent coarse-grained model of double-stranded DNA (dsDNA), where each nucleotide is described by three beads localized at the sugar, at the base moiety, and at the phosphate group, respectively. The sequence dependence is included by considering a step-dependent parametrization of the bonded interactions, which are tuned in order to reproduce the values of key observables obtained from exhaustive atomistic simulations from the literature. The predictions of the model are benchmarked against an independent set of all-atom simulations, showing that it captures with high fidelity the sequence dependence of conformational and elastic features beyond the single step considered in its formulation. A remarkably good agreement with experiments is found for both sequence-averaged and sequence-dependent conformational and elastic features, including the stretching and torsion moduli, the twist-stretch and twist-bend couplings, the persistence length, and the helical pitch. Overall, for the inspected quantities, the model has a precision comparable to atomistic simulations, hence providing a reliable coarse-grained description for the rationalization of single-molecule experiments and the study of cellular processes involving dsDNA. Owing to the simplicity of its formulation, MADna can be straightforwardly included in common simulation engines. Particularly, an implementation of the model in LAMMPS is made available on an online repository to ease its usage within the DNA research community.

Enzymatic hydrolysis of monoacylglycerols and their cyclopropanated derivatives: Molecular structure and nanostructure determine the rate of digestion.

Colloidal lipidic particles with different space groups and geometries (mesosomes) are employed in the development of new nanosystems for the oral delivery of drugs and nutrients. Understanding of the enzymatic digestion rate of these particles is key to the development of novel formulations. In this work, the molecular structure of the lipids has been systematically tuned to examine the effect on their self-assembly and digestion rate. The kinetic and phase changes during the lipase-catalysed hydrolysis of mesosomes formed by four synthetic cyclopropanated lipids and their cis-unsaturated analogues were monitored by dynamic small angle X-ray scattering and acid/base titration. It was established that both the phase behaviour and kinetics of the hydrolysis are greatly affected by small changes in the molecular structure of the lipid as well as by the internal nanostructure of the colloidal particles.

Interplay between Confinement and Drag Forces Determine the Fate of Amyloid Fibrils.

The fine interplay between the simultaneous stretching and confinement of amyloid fibrils is probed by combining a microcapillary setup with atomic force microscopy. Single-molecule statistics reveal how the stretching of fibrils changed from force to confinement dominated at different length scales. System order, however, is solely ruled by confinement. Coarse-grained simulations support the results and display the potential to tailor system properties by tuning the two effects. These findings may further help shed light on in vivo amyloid fibril growth and transport in highly confined environments such as blood vessels.

Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones.

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

Six-fold director field configuration in amyloid nematic and cholesteric phases.

Chiral liquid crystals, or cholesteric phases, have been widely studied in the last decades, leading to fundamental advances and a multitude of applications and technologies. In general, the rich phenomenology of these systems depends directly on the molecular traits and conditions of the system, imposing precise symmetry to the resulting nematic field. By selecting amyloid fibrils as model filamentous chiral colloids, we report an unprecedented breadth of liquid crystalline morphologies, where up to six distinct configurations of the nematic field are observed under identical conditions. Amyloid-rich droplets show homogeneous, bipolar, radial, uniaxial chiral and radial chiral nematic fields, with additional parabolic focal conics in bulk. Variational and scaling theories allow rationalizing the experimental evidence as a subtle interplay between surface and bulk energies. Our experimental and theoretical findings deepen the understanding of chiral liquid crystals under confinement, opening to a more comprehensive exploitation of these systems in related functional materials.

Soft biomimetic nanoconfinement promotes amorphous water over ice.

Water is a ubiquitous liquid with unique physicochemical properties, whose nature has shaped our planet and life as we know it. Water in restricted geometries has different properties than in bulk. Confinement can prevent low-temperature crystallization of the molecules into a hexagonal structure and thus create a state of amorphous water. To understand the survival of life at subzero temperatures, it is essential to elucidate this behaviour in the presence of nanoconfining lipidic membranes. Here we introduce a family of synthetic lipids with designed cyclopropyl modifications in the hydrophobic chains that exhibit unique liquid-crystalline behaviour at low temperature, which enables the maintenance of amorphous water down to ~10 K due to nanoconfinement. The combination of experiments and molecular dynamics simulations unveils a complex lipid-water phase diagram in which bicontinuous cubic and lamellar liquid crystalline phases that contain subzero liquid, glassy or ice water emerge as a competition between the two components, each pushing towards its thermodynamically favoured state.

Spatiotemporal Control of Enzyme-Induced Crystallization Under Lyotropic Liquid Crystal Nanoconfinement.

Water nanoconfinement has important effects on the properties of biomolecules and ultimately on their specific functions. By performing experiments and molecular dynamic simulations, we show how intrinsic nanoconfinement controls the crystallization of small organic molecules converted by enzymatic reactions within the water nanochannels of lipid cubic phases (LCPs). By controlling the nanochannel size, enzymatic reactions in LCPs can be engineered to turn the same converted substrate into its soluble, microcrystal, or needle-like crystal form due to the large variability in water dynamics. Differential scanning calorimetry studies, supported by molecular dynamics simulations, show that most of water within the mesophase nanochannels behaves differently due to interactions with the LCP interface, and that this mechanism has a larger impact for smaller channels. These findings suggest that the amount of free water in the core of the nanochannels is the key factor determining local substrate diffusion and self-assembly within LCPs.

Impact of Molecular Partitioning and Partial Equilibration on the Estimation of Diffusion Coefficients from Release Experiments.

The present work addresses the effect of partial equilibration and molecular partitioning on the interpretation of release experiments. In this regard, it is shown how release profiles and the values of extracted transport parameters are affected by the time protocol chosen for sample collection by considering a series of experiments where the latter is systematically varied. Caffeine is investigated as a main model drug because of its similar affinity for water and lipids, while monolinolein-based lipid cubic phases are chosen as host matrices because of their wide employment in release studies. Our findings point to a progressive decline in diffusion rate upon increasing the time step, that is, the gap in time between two consecutive pickups, which is a signature of increasing equilibration of caffeine concentration between the lipidic mesophase and the water phase. Furthermore, the amount of released molecules at the first pickup displays negligible changes for large time steps, indicating complete equilibration in such cases. A model is introduced based on Fick's diffusion which goes beyond the assumption of perfect-sink conditions, a common feature of the typical theoretical approaches hitherto developed. The model is shown to account quantitatively for the experimental data and is subsequently employed to clarify the interplay of the adopted release protocol with the various transport parameters in determining the final outcome of the release process. Particularly, two additional molecular drugs are considered, namely glucose and proflavine, which are, respectively, more hydrophilic and hydrophobic than caffeine, thus allowing elucidating the role of molecular partitioning.

Confinement-Induced Ordering and Self-Folding of Cellulose Nanofibrils.

Cellulose is a pervasive polymer, displaying hierarchical lengthscales and exceptional strength and stiffness. Cellulose's complex organization, however, also hinders the detailed understanding of the assembly, mesoscopic properties, and structure of individual cellulose building blocks. This study combines nanolithography with atomic force microscopy to unveil the properties and structure of single cellulose nanofibrils under weak geometrical confinement. By statistical analysis of the fibril morphology, it emerges that confinement induces both orientational ordering and self-folding of the fibrils. Excluded volume simulations reveal that this effect does not arise from a fibril population bias applied by the confining slit, but rather that the fibril conformation itself changes under confinement, with self-folding favoring fibril's free volume entropy. Moreover, a nonstochastics angular bending probability of the fibril kinks is measured, ruling out alternating amorphous-crystalline regions. These findings push forward the understanding of cellulose nanofibrils and may inspire the design of functional materials based on fibrous templates.

The interplay of channel geometry and molecular features determines diffusion in lipidic cubic phases.

The transport behavior of inverse bicontinuous cubic phases is experimentally investigated as the combined outcome of solute molecular structure and geometrical details of the confining symmetry. Molecular diffusion is discussed in relation to curvature, bottlenecks, and interfacial properties of each cubic phase. Point-like molecules show faster diffusion across the double diamond (Pn3¯m) symmetry, while unfolded macromolecules display better performance inside the double primitive (Im3¯m) cubic phase. The former observation is in agreement with previous simulation work, whereas the latter indicates that dedicated theory needs to be developed for diffusing polymers. Furthermore, the effect of electrostatic interactions is assessed by a study of diffusion of nanoparticles and is rationalized via a combination of simulations and theoretical considerations as the result of a competition between water mobility and geometrical features of the channel.

Curvature and bottlenecks control molecular transport in inverse bicontinuous cubic phases.

We perform a simulation study of the diffusion of small solutes in the confined domains imposed by inverse bicontinuous cubic phases for the primitive, diamond, and gyroid symmetries common to many lipid/water mesophase systems employed in experiments. For large diffusing domains, the long-time diffusion coefficient shows universal features when the size of the confining domain is renormalized by the Gaussian curvature of the triply periodic minimal surface. When bottlenecks are widely present, they become the most relevant factor for transport, regardless of the connectivity of the cubic phase.

Shape of a Stretched Polymer.

The shape of a polymer plays an important role in its interactions with surrounding molecules. We characterize the shape and the orientational properties of a polymer chain under tension in a good solvent, a physical condition that is often realized both in single-molecule experiments and in vivo. Our findings reveal the existence of hitherto unobserved universal laws encompassing polymers with different rigidities and including the possible presence of excluded-volume effects, showing that both shape and orientation are solely determined by the force contribution to the free energy. In doing so, they also provide a simple way to retrieve these quantities from the knowledge of the force-versus-extension curve.