Insight into the Molecular Mechanisms of Protein Stabilizing Osmolytes from Global Force-Field Variations

A prominent class of osmolytes that are able to stabilize proteins in their native fold consist of small highly water-soluble molecules with a large dipole moment and hydrophobic groups attached to the positively charged end of the molecule, for which we coin the term dipolar/hydrophobic osmolytes. For TMAO, which is a prime member of this class, we perform large-scale water-explicit MD simulations and determine the bulk solution activity coefficient as well as the affinity to a stretched polyglycine chain for varying TMAO dipolar strength and hydrophobicity. Double optimization with respect to experimental values for the activity coefficient and the polyglycine transfer free energy is achieved. The resulting optimal TMAO force field shows excellent transferability to different concentrations and also reproduces transfer free energies of various amino acids, including the tryptophan anomaly, for which TMAO acts as a denaturant. By globally analyzing the thermodynamic and structural properties of suboptimal TMAO force fields, we identify the frustration between dipolar and hydrophobic interactions as the working mechanism and the design principle of dipolar/hydrophobic osmolytes

The Complete Phase Diagram of Monolayers of Enantiomeric <i>N</i>‑Stearoyl-threonine Mixtures with Preferred Heterochiral Interactions

Langmuir monolayers of chiral amphiphiles are well-controlled model systems for the investigation of phenomena related to stereochemistry. Here, we have investigated mixed monolayers of one pair of enantiomers (l and d) of the amino-acid-based amphiphile N-stearoyl-threonine. The monolayer characteristics were studied by pressure–area isotherm measurements and grazing incidence X-ray diffraction (GIXD) over a wide range of mixing ratios defined by the d-enantiomer mole fraction xD. While the isotherms provide insights into thermodynamical aspects, such as transition pressure, compression/decompression hysteresis, and preferential homo- and heterochiral interactions, GIXD reveals the molecular structural arrangements on the Ångström scale. Dominant heterochiral interactions in the racemic mixture lead to compound formation and the appearance of a nonchiral rectangular lattice, although the pure enantiomers form a chiral oblique lattice. Miscibility was found to be limited to mixtures with 0.27 ≲ xD ≲ 0.73, as well as to both outer edges (xD ≲ 0.08 and xD ≳ 0.92). Beyond this range, coexistence of oblique and rectangular lattices occurs, as is clearly seen in the GIXD patterns. Based on the results, a complete phase diagram with two eutectic points at xD ≈ 0.25 and xD ≈ 0.75 is proposed. Moreover, N-stearoyl-threonine was found to have a strong tendency to form a hydrogen-bonding network between the headgroups, which promotes superlattice formation

Grazing-Incidence Neutron-Induced Fluorescence Probes Density Profiles of Labeled Molecules at Solid/Liquid Interfaces

We report on the use of characteristic prompt γ-fluorescence after neutron capture induced by an evanescent neutron wave to probe densities and depth profiles of labeled molecules at solid/liquid interfaces. In contrast to classical scattering techniques and X-ray fluorescence, this method of “grazing-incidence neutron-induced fluorescence” combines direct chemical specificity, provided by the label, with sensitivity to the interface, inherent to the evanescent wave. We demonstrate that the formation of a supported lipid membrane can be quantitatively monitored from the characteristic fluorescence of ¹⁵⁷Gd³⁺ ions bound to the headgroup of chelator lipids. Moreover, we were able to localize the ¹⁵⁷Gd³⁺ ions along the surface normal with nanometer precision. This first proof of principle with a well-defined model system suggests that the method has a great potential for biology and soft matter studies where spatial resolution and chemical sensitivity are required

Generic Role of Polymer Supports in the Fine Adjustment of Interfacial Interactions between Solid Substrates and Model Cell Membranes

To understand the generic role of soft, hydrated biopolymers in adjusting interfacial interactions at biological interfaces, we designed a defined model of the cell–extracellular matrix contacts based on planar lipid membranes deposited on polymer supports (polymer-supported membranes). Highly uniform polymer supports made out of regenerated cellulose allow for the control of film thickness without changing the surface roughness and without osmotic dehydration. The complementary combination of specular neutron reflectivity and high-energy specular X-ray reflectivity yields the equilibrium membrane–substrate distances, which can quantitatively be modeled by computing the interplay of van der Waals interaction, hydration repulsion, and repulsion caused by the thermal undulation of membranes. The obtained results help to understand the role of a biopolymer in the interfacial interactions of cell membranes from a physical point of view and also open a large potential to generally bridge soft, biological matter and hard inorganic materials

Water Structuring Induces Nonuniversal Hydration Repulsion between Polar Surfaces: Quantitative Comparison between Molecular Simulations, Theory, and Experiments

Polar surfaces in water typically repel each other at close separations, even if they are charge-neutral. This so-called hydration repulsion balances the van der Waals attraction and gives rise to a stable nanometric water layer between the polar surfaces. The resulting hydration water layer is crucial for the properties of concentrated suspensions of lipid membranes and hydrophilic particles in biology and technology, but its origin is unclear. It has been suggested that surface-induced molecular water structuring is responsible for the hydration repulsion, but a quantitative proof of this water-structuring hypothesis is missing. To gain an understanding of the mechanism causing hydration repulsion, we perform molecular simulations of different planar polar surfaces in water. Our simulated hydration forces between phospholipid bilayers agree perfectly with experiments, validating the simulation model and methods. For the comparison with theory, it is important to split the simulated total surface interaction force into a direct contribution from surface–surface molecular interactions and an indirect water-mediated contribution. We find the indirect hydration force and the structural water-ordering profiles from the simulations to be in perfect agreement with the predictions from theoretical models that account for the surface-induced water ordering, which strongly supports the water-structuring hypothesis for the hydration force. However, the comparison between the simulations for polar surfaces with different headgroup architectures reveals significantly different decay lengths of the indirect water-mediated hydration-force, which for laterally homogeneous water structuring would imply different bulk-water properties. We conclude that laterally inhomogeneous water ordering, induced by laterally inhomogeneous surface structures, shapes the hydration repulsion between polar surfaces in a decisive manner. Thus, the indirect water-mediated part of the hydration repulsion is caused by surface-induced water structuring but is surface-specific and thus nonuniversal

Combination of MD Simulations with Two-State Kinetic Rate Modeling Elucidates the Chain Melting Transition of Phospholipid Bilayers for Different Hydration Levels

The phase behavior of membrane lipids plays an important role in the formation of functional domains in biological membranes and crucially affects molecular transport through lipid layers, for instance, in the skin. We investigate the thermotropic chain melting transition from the ordered <i>L</i>_β phase to the disordered <i>L</i>_α phase in membranes composed of dipalmitoylphosphatidylcholine (DPPC) by atomistic molecular dynamics simulations in which the membranes are subject to variable heating rates. We find that the transition is initiated by a localized nucleus and followed by the propagation of the phase boundary. A two-state kinetic rate model allows characterizing the transition state in terms of thermodynamic quantities such as transition state enthalpy and entropy. The extrapolated equilibrium melting temperature increases with reduced membrane hydration and thus in tendency reproduces the experimentally observed dependence on dehydrating osmotic stress

Neutron Reflectometry Elucidates Protein Adsorption from Human Blood Serum onto PEG Brushes

Poly­(ethylene glycol) (PEG) brushes are reputed for their ability to prevent undesired protein adsorption to material surfaces exposed to biological fluids. Here, protein adsorption out of human blood serum onto PEG brushes anchored to solid-supported lipid monolayers was characterized by neutron reflectometry, yielding volume fraction profiles of lipid headgroups, PEG, and adsorbed proteins at subnanometer resolution. For both PEGylated and non-PEGylated lipid surfaces, serum proteins adsorb as a thin layer of approximately 10 Å, overlapping with the headgroup region. This layer corresponds to primary adsorption at the grafting surface and resists rinsing. A second diffuse protein layer overlaps with the periphery of the PEG brush and is attributed to ternary adsorption due to protein–PEG attraction. This second layer disappears upon rinsing, thus providing a first observation of the structural effect of rinsing on protein adsorption to PEG brushes

DSC thermograms of myelin (20 Mm) under different aqueous media.

<p>In the cases where homogenization takes place–bi-distilled water (black line) and near physiological medium (red line)–a maximum is reached after which a stabilization of the C_p is observed near physiological temperature. In the case of high [Ca²⁺] (blue line), a continuous variation of C_p according to a phase redistribution of components is observed.</p

Number (<i>n</i>) of correlated membranes for the different phases as a function of temperature for whole myelin (empty) and DIGs (filled).

<p>In black is marked the <i>n</i> for the native period, in red for the expanded period and in blue for the compact period. In the presence of physiological (diamonds) and high [Ca²⁺] (circles) media.</p

Neutron diffraction (planar system) periodicities compared to SAXS on equivalent conditions.

<p>Neutron diffraction (planar system) periodicities compared to SAXS on equivalent conditions.</p