Metastable precipitation and ion-extractant transport in liquid-liquid separations of trivalent elements.

The extractant-assisted transport of metal ions from aqueous to organic environments by liquid-liquid extraction has been widely used to separate and recover critical elements on an industrial scale. While current efforts focus on designing better extractants and optimizing process conditions, the mechanism that underlies ionic transport remains poorly understood. Here, we report a nonequilibrium process in the bulk aqueous phase that influences interfacial ion transport: the formation of metastable ion-extractant precipitates away from the liquid-liquid interface, separated from it by a depletion region without precipitates. Although the precipitate is soluble in the organic phase, the depletion region separates the two and ions are sequestered in a long-lived metastable state. Since precipitation removes extractants from the aqueous phase, even extractants that are sparingly soluble in water will continue to be withdrawn from the organic phase to feed the aqueous precipitation process. Solute concentrations in both phases and the aqueous pH influence the temporal evolution of the process and ionic partitioning between the precipitate and organic phase. Aqueous ion-extractant precipitation during liquid-liquid extraction provides a reaction path that can influence the extraction kinetics, which plays an important role in designing advanced processes to separate rare earths and other minerals.

MFG-E8: a model of multiple binding modes associated with ps-binding proteins.

Membrane-binding proteins often associate with lipid membranes through a singular binding interface which is generally modeled as a two-state system: bound or unbound. However, even a single interface can engage with more than one mode of binding since a variety of interactions can contribute to the binding event. Unfortunately, the ability to clearly delineate the different binding modes of a singular binding interface has been elusive with existing models. Here, we present a study on milk fat globule EGF factor 8 (MFG-E8), which belongs to a class of proteins that identifies and binds phosphatidylserine (PS). These proteins detect membrane dysregulation implicated in exposed PS in apoptosis and malignant cells. In order to elucidate the factors affecting the binding of MFG-E8, we used a model system consisting of a series of lipid vesicles with varying PS mole fraction to identify the sensitivity of MFG-E8's binding affinity to changes in electrostatics using a tryptophan fluorescence spectral shift assay. Using a newly developed model, we experimentally identified three binding modes, each associated with a different number of PS lipids, with its cooperativity for binding being enhanced by the availability of negatively charged lipids. X-ray reflectivity experiments additionally suggest that MFG-E8's binding modes are influenced by membrane packing. The protocols established for elucidating MFG-E8's interaction with lipid membranes under different membrane conditions can be applied to the study of other membrane-binding proteins that target specific membrane attributes, such as fluidity and electrostatics, and help elucidate these membrane targeting mechanisms and their subsequent binding events.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

Free Thiols Regulate the Interactions and Self-Assembly of Thiol-Passivated Metal Nanoparticles.

Thiol ligands bound to the metallic core of nanoparticles determine their interactions with the environment and self-assembly. Recent studies suggest that equilibrium between bound and free thiols alters the ligand coverage of the core. Here, X-ray scattering and MD simulations investigate water-supported monolayers of gold-core nanoparticles as a function of the core-ligand coverage that is varied in experiments by adjusting the concentration of total thiols (sum of free and bound thiols). Simulations demonstrate that the presence of free thiols produces a nearly symmetrical coating of ligands on the core. X-ray measurements show that above a critical value of core-ligand coverage the nanoparticle core rises above the water surface, the edge-to-edge distance between neighboring nanoparticles increases, and the nanoparticle coverage of the surface decreases. These results demonstrate the important role of free thiols: they regulate the organization of bound thiols on the core and the interactions of nanoparticles with their surroundings.

How Tim proteins differentially exploit membrane features to attain robust target sensitivity.

Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

Knowledge-Based Design of 5-Fluororacil Prodrug Liposomal Formulation: Molecular Packing and Interaction Revealed by Interfacial Isotherms and X-ray Scattering Techniques.

Prodrugs and nanoformulations are two effective strategies for sustained drug release and targeting drug delivery. In this study, we combined the two strategies to judiciously design the liposome formulation incorporating an amphiphilic prodrug of 5-fouroracil (5-FU), named 5-FCPal, for sustained drug release and enhanced bioavailability. 5-FCPal is an analogue of capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine, Xeloda) by substituting the pentyl group at the N4 position with the palmityl. The amphiphilic molecule of 5-FCPal can self-assemble with the phospholipids to form stable vesicle structures with high drug loading. Although lipid vesicles have been widely studied and commercially used for clinical applications, because of the enormous options of the lipids and the equitable balance of hydrophobicity and bioavailability, it is essential to fundamentally understand the molecular interactions when designing and optimizing the liposomal prodrug formulations. We report the study of using X-ray liquid surface scattering techniques integrated with a Langmuir trough to explicitly reveal the interfacial behavior of the monolayer membrane of 5-FCPal with various saturated and unsaturated lipids with positively charged, neutral, and negatively charged head groups. More specifically, interfacial packing of the molecules was quantified using interfacial isotherms, X-ray reflectivity (XR), and grazing-incidence diffraction (GIXD). The results indicate that the interactions between the prodrug and the cationic lipids are most favorable. The highest drug loading is quantified by increasing the molar ratio of the prodrug until stable monolayer structures were disrupted by the multiple-layer domain of prodrug aggregates. Stable liposomes of 100 nm with 50% drug loading of 5-FCPal were generated based on the findings from the X-ray studies.

Influence of Substitutional Groups on the Ordering and Crystallization of Amphiphilic Silsesquioxanes at the Air-Water Interface.

We report on the surface ordering and crystallization sequences in differently organic-substituted amphiphilic polyhedral silsesquioxane (POSS) variants induced by regulated compression at the air-water interface. Such molecular systems floating at the interface serve as a model system to study dynamic crystallization mediated by weak interactions. In situ grazing incidence X-ray scattering (GIXS) measurements, performed at a synchrotron X-ray source using a liquid surface diffractometer and corroborated with Brewster angle microscopy, revealed transformations for the different POSS variants (viz. trisilanol isobutyl POSS (TBPOSS), trisilanol cyclohexyl POSS (TCHPOSS), disilanol octaisobutly POSS (DOBPOSS), and trisilanol isooctyl POSS (TOPOSS)) from a weakly correlated monolayer structure to appreciably different structural and crystalline phases in various packing schemes. GIXS measurements revealed a stable nature of the crystallization of DOBPOSS, varying degrees of metastable crystallization for TCHPOSS and TBPOSS, and complete absence of crystalline phase in TOPOSS molecules. Incidentally, for all POSS variants showing crystalline phases, the motifs always assembled in a triclinic lattice with P1̅ symmetry. For the metastable crystals, preferential surface ordering of the crystallites promotes selective crystalline planes to exhibit preferred tilt angles with respect to the interface. The structural transformations of the differently substituted POSS molecules and their variations therein are attributed to the changing balance of the hydrophobic vs hydrophilic interaction in the layers, which is determined by the anisotropic shape and distribution of substitutional groups over the nanosized core cage of the monomer, steric interaction between nearest dimeric neighbors, as well as the in-plane and out-of-plane assembly of the overlayers.

Interaction between dilute water vapor and dodecane thiol ligated Au nanoparticles: Hydrated structure and pair potential of mean force.

The interaction between two ligated nanoparticles depends on whether they are isolated or immersed in a liquid solvent. However, very little is known about the influence of solvent vapor on the interaction between two ligated nanoparticles. Recent experiments yield the surprising result that the cyclic exposure of solvent free suspended monolayers of dodecane thiol ligated gold nanoparticles (AuNPs) to water vapor and dry nitrogen generates reversible cyclic decreases and increases in Young's modulus of the monolayer, implying corresponding cyclic changes in the AuNP-AuNP interaction. We examine how water vapor interacts with an isolated dodecane thiol dressed AuNP and how water vapor affects the interaction between a pair of nanoparticles, using all-atom molecular-dynamics simulations. We find that there is condensation of water molecules onto the ligand shell of an AuNP in the form of clusters of 100-2000 molecules that partially cover the shell, with most of the water in a few large clusters. A water cluster bridges the AuNPs, with a sensibly constant number of water molecules for AuNP-AuNP separations from the edge-to-edge contact up to center-to-center separations of 100 Å. The wet AuNP-AuNP interaction has a slightly deeper and wider asymmetric well than does the dry interaction, a change that is qualitatively consistent with that implied by the observed water vapor induced change in Young's modulus of a monolayer of these AuNPs. We find that macroscopic analyses of water drop-deformable surface interactions and dynamics provide both guidance to understanding and qualitatively correct predictions of the phenomena observed in our simulations.

Impeded Molecular Reorganization by Polyethylene Glycol Conjugation Revealed by X-ray Reflectivity and Diffraction Measurements.

Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas-liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid-Ca2+ complexes.

Structure and dynamics of lipid membranes interacting with antivirulence end-phosphorylated polyethylene glycol block copolymers.

The structure and dynamics of lipid membranes in the presence of extracellular macromolecules are critical for cell membrane functions and many pharmaceutical applications. The pathogen virulence-suppressing end-phosphorylated polyethylene glycol (PEG) triblock copolymer (Pi-ABAPEG) markedly changes the interactions with lipid vesicle membranes and prevents PEG-induced vesicle phase separation in contrast to the unphosphorylated copolymer (ABAPEG). Pi-ABAPEG weakly absorbs on the surface of lipid vesicle membranes and slightly changes the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar vesicles at 37 °C, as evidenced by small angle neutron scattering. X-ray reflectivity measurements confirm the weak adsorption of Pi-ABAPEG on DMPC monolayer, resulting in a more compact DMPC monolayer structure. Neutron spin-echo results show that the adsorption of Pi-ABAPEG on DMPC vesicle membranes increases the membrane bending modulus κ.

Single-pixel interior filling function approach for detecting and correcting errors in particle tracking.

We present a general method for detecting and correcting biases in the outputs of particle-tracking experiments. Our approach is based on the histogram of estimated positions within pixels, which we term the single-pixel interior filling function (SPIFF). We use the deviation of the SPIFF from a uniform distribution to test the veracity of tracking analyses from different algorithms. Unbiased SPIFFs correspond to uniform pixel filling, whereas biased ones exhibit pixel locking, in which the estimated particle positions concentrate toward the centers of pixels. Although pixel locking is a well-known phenomenon, we go beyond existing methods to show how the SPIFF can be used to correct errors. The key is that the SPIFF aggregates statistical information from many single-particle images and localizations that are gathered over time or across an ensemble, and this information augments the single-particle data. We explicitly consider two cases that give rise to significant errors in estimated particle locations: undersampling the point spread function due to small emitter size and intensity overlap of proximal objects. In these situations, we show how errors in positions can be corrected essentially completely with little added computational cost. Additional situations and applications to experimental data are explored in SI Appendix In the presence of experimental-like shot noise, the precision of the SPIFF-based correction achieves (and can even exceed) the unbiased Cramér-Rao lower bound. We expect the SPIFF approach to be useful in a wide range of localization applications, including single-molecule imaging and particle tracking, in fields ranging from biology to materials science to astronomy.

Impact of Surface Amphiphilicity on the Interfacial Behavior of Janus Particle Layers under Compression.

Langmuir monolayers of silica/gold Janus particles with two different degrees of amphiphilicity have been examined to study the significance of particle surface amphiphilicity on the structure and mechanical properties of the interfacial layers. The response of the layers to the applied compression provides insight into the nature and strength of the interparticle interactions. Different collapse modes observed for the interfacial layers are linked to the amphiphilicity of Janus particles and their configuration at the interface. Molecular dynamics simulations on nanoparticles with similar contact angles provide insight on the arrangement of particles at the interface and support our conclusion that the interfacial configuration and collapse of anisotropic particles at the air/water interface are controlled by particle amphiphilicity.

Polyunsaturated Phospholipid Modified Membrane Degradation Catalyzed by a Secreted Phospholipase A2.

To optimize the compositions of the lipid-based nanomedicine and to advance understanding of the roles of polyunsaturated phospholipids in biological membranes, this study examined the effects of polyunsaturated phospholipids on the degradation of giant unilamellar vesicles catalyzed by a secreted phospholipase A2 (sPLA2) using fluorescence microscopy. Molecular interfacial packing, interaction, and degradation of the films containing various mixing ratios of saturated and polyunsaturated phospholipids were quantified using a Langmuir trough integrated with synchrotron X-ray surface scattering techniques. It was found that a high molar fraction (0.63 and above) of polyunsaturated phospholipids not only enhanced the rate of sPLA2-catalyzed vesicle degradation but also changed the vesicle deformation process and degradation product morphology. Hydrolysis of the saturated phospholipids generated highly ordered liquid crystal domains, which was reduced or prohibited by the presence of the polyunsaturated phospholipids in the reactant film.

Tailoring Biomimetic Phosphorylcholine-Containing Block Copolymers as Membrane-Targeting Cellular Rescue Agents.

Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.

The influence of fractional surface coverage on the core-core separation in ordered monolayers of thiol-ligated Au nanoparticles.

We report the results of grazing incidence X-ray diffraction (GIXD) measurements from water supported Langmuir monolayers of gold nanoparticles ligated with dodecanethiol (12 carbons), tetradecanethiol (14 carbons), hexadecanethiol (16 carbons), and octadecanethiol (18 carbons). These monolayers are formed from solutions with varying concentrations of the respective thiols. We show that equilibrium between adsorbed thiol molecules and the thiols in the bulk solution implies fractional coverage of the Au nanoparticle core. We also show that the nanoparticle-nanoparticle separation and the correlation length of particles in these ordered films increases with thiol concentration in the parent solution, and that excess thiol can be found in the space between particles as well as in islands away from the particles. Using the equilibrium constant relating ligand solution concentration and nanoparticle surface coverage of the gold core by the ligand molecules, we interpret the way in which varying thiol concentration affects the nanoparticle-nanoparticle separation as a function of surface coverage of the gold core by the ligand molecules.

Molecular interactions of phospholipid monolayers with a model phospholipase.

The intrinsic overexpression of secretory phospholipase A2 (sPLA2) in various pro-inflammatory diseases and cancers has the potential to be exploited as a therapeutic strategy for diagnostics and treatment. To explore this potential and advance our knowledge of the role of sPLA2 in related diseases, it is necessary to systematically investigate the molecular interaction of the enzyme with lipids. By employing a Langmuir trough integrated with X-ray reflectivity and grazing incidence X-ray diffraction techniques, this study examined the molecular packing structure of 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) films before and after enzyme adsorption and enzyme-catalyzed degradation. Molecular interaction of sPLA2 (from bee venom) with the DPPC monolayer exhibited Ca2+ dependence. DPPC molecules at the interface without Ca2+ retained a monolayer organization; upon adsorption of sPLA2 to the monolayer the packing became tighter. In contrast, sPLA2-catalyzed degradation of DPPC occurred in the presence of Ca2+, leading to disruption of the ordered monolayer structure of DPPC. The interfacial film became a mixture of highly ordered multilayer domains of palmitic acid (PA) and loosely packed monolayer phase of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC) that potentially contained the remaining un-degraded DPPC. The redistribution of lipid degradation products into the third dimension, which produced multilayer PA domains, damaged the structural integrity of the original lipid layer and may explain the bursting of liposomes observed in other studies after a latency period of mixing liposomes with sPLA2. A quantitative understanding of the lipid packing and lipid-enzyme interaction provides an intuitive means of designing and optimizing lipid-related drug delivery systems.

Pair and many-body interactions between ligated Au nanoparticles.

We report the results of molecular dynamics simulations of the properties of a pseudo-atom (united atom) model of dodecane thiol ligated 5-nm diameter gold nanoparticles (AuNPs) in a vacuum as a function of ligand coverage and particle separation in three states of aggregation, namely, the isolated AuNPs, the isolated pair of AuNPs, and a square lattice of four AuNPs. Our calculations show that the ligand density along a radius emanating from the core of an isolated AuNP has the same gross features for all values of the coverage; it oscillates around a constant value up to a distance along the chain corresponding to the position of the fourth pseudo-atom and then smoothly decays to zero, reflecting both the restricted conformations of the chain near the core surface and the larger numbers of conformations available further from the core. Interaction between two AuNPs generates changes in the ligand distributions of each. We examine the structure and general shape of the ligand envelope as a function of the coverage and demonstrate that the equilibrium structure of the envelope and the deformation of that envelope generated by interaction between the NPs are coverage-dependent so that the shape, depth, and position of the minimum of the potential of mean force display a systematic dependence on the ligand coverage. We propose an accurate analytical description of the calculated potential of mean force as a function of a set of parameters that scale linearly with the ligand coverage. Noting that the conformational freedom of the ligands implies that multiparticle induced deviations from additivity of the pair potential of mean force are likely important; we define and calculate a "bond stretching" effective pair potential of mean force for a square lattice of particles that contains, implicitly, both the three- and four-NP contributions. We find that the bond stretching effective pair potential of mean force in this cluster has a different minimum and a different well depth from the isolated pair potential of mean force. Previous work has found that the three-particle contribution to deviation from pair additivity is monotonically repulsive, whereas we find that the combined three- and four-particle contributions have an attractive well, implying that the three- and four-particle contributions are of comparable magnitude but opposite sign, thereby suggesting that even higher order correction terms likely play a significant role in the behavior of dense assemblies of many nanoparticles.

A comment on the position dependent diffusion coefficient representation of structural heterogeneity.

Experimental studies of the variation of the mean square displacement (MSD) of a particle in a confined colloid suspension that exhibits density variations on the scale length of the particle diameter are not in agreement with the prediction that the spatial variation in MSD should mimic the spatial variation in density. The predicted behavior is derived from the expectation that the MSD of a particle depends on the system density and the assumption that the force acting on a particle is a point function of position. The experimental data are obtained from studies of the MSDs of particles in narrow ribbon channels and between narrowly spaced parallel plates and from new data, reported herein, of the radial and azimuthal MSDs of a colloid particle in a dense colloid suspension confined to a small circular cavity. In each of these geometries, a dense colloid suspension exhibits pronounced density oscillations with spacing of a particle diameter. We remove the discrepancy between prediction and experiment using the Fisher-Methfessel interpretation of how local equilibrium in an inhomogeneous system is maintained to argue that the force acting on a particle is delocalized over a volume with radius equal to a particle diameter. Our interpretation has relevance to the relationship between the scale of inhomogeneity and the utility of translation of the particle MSD into a position dependent diffusion coefficient and to the use of a spatially dependent diffusion coefficient to describe mass transport in a heterogeneous system.

Transient structured fluctuations in a two-dimensional system with multiple ordered phases.

We report the structure of transient fluctuations in the liquid phase of a two-dimensional system that exhibits several ordered phases with different symmetries. The density-temperature phase diagram of the system studied, composed of particles with a repulsive shouldered soft-core pair interaction, has regions with stable liquid and hexatic phases, a square solid phase, two separate hexagonal solid phases, and a quasi-crystalline phase with 12-fold symmetry. We have examined the character of the structured fluctuations by computing the same-time aperture cross correlation function of particle configurations in several fluid regions near to and far from phase transition lines. The two primary goals of our study are (1) determination if the spectrum of structures of the fluctuations in the liquid is broader than or limited to the motifs exhibited by the ordered phases supported by the system and (2) determination of the density domains in the liquid that support particular transient structured fluctuations. In the system studied, along a low-temperature isotherm in the temperature-density plane that intersects all the ordered phases we find that the liquid phase exhibits structured fluctuations with hexagonal symmetry near both liquid-hexatic transition lines. Along the same isotherm and in the stable liquid between the lower density hexatic-to-liquid and the higher density liquid-to-square solid transitions, we find that transient hexagonal ordered fluctuations dominate the liquid region near the hexatic-to-liquid transition and square ordered fluctuations dominate the liquid region near the liquid-to square solid transition, but both of these structured fluctuations occur at all densities between these transition lines. At a higher temperature, at phase points in the liquid above, but close to the density maximum of an underlying transition, there are ordered fluctuations that can be correlated with the structure of the lower temperature phase. Although it is expected that very close to a liquid-ordered phase boundary a structured fluctuation in the liquid will have the same symmetry as the ordered phase, it is not obvious that structured fluctuations in thermodynamic states deep in the liquid phase will be similarly restricted. The most striking result of our calculations is that no evidence is found in the liquid phase for structured fluctuations with other symmetries than those of the ordered phases of the system.

Coupling X-Ray Reflectivity and In Silico Binding to Yield Dynamics of Membrane Recognition by Tim1.

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.