Trimethylamine-N-oxide depletes urea in a peptide solvation shell.

Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.

PDMS as a Substrate for Lipid Bilayers.

PDMS (polydimethylsiloxane) is a cheap, optically clear polymer that is elastic and can be easily and quickly fabricated into a wide array of microscale and nanoscale architectures, making it a versatile substrate for biophysical experiments on cell membranes. It is easy to imagine many new experiments will be devised that require a bilayer to be placed upon a substrate that is flexible or easily cast into a desired geometry, such as in lab-on-a-chip, organ-on-chip, and microfluidic applications, or for building accurate membrane models that replicate the surface structure and elasticity of the cytoskeleton. However, PDMS has its limitations, and the extent to which the behavior of membranes is affected on PDMS has not been fully explored. We use AFM and fluorescence optical microscopy to investigate the use of PDMS as a substrate for the formation and study of supported lipid bilayers (SLBs). Lipid bilayers form on plasma-treated PDMS and show free diffusion and normal phase transitions, confirming its suitability as a model bilayer substrate. However, lipid-phase separation on PDMS is severely restricted due to the pinning of domains to surface roughness, resulting in the cessation of lateral hydrodynamic flow. We show the high-resolution porous structure of PDMS and the extreme smoothing effect of oxygen plasma treatment used to hydrophilize the surface, but this is not flat enough to allow domain formation. We also observe bilayer degradation over hour timescales, which correlates with the known hydrophobic recovery of PDMS, and establish a critical water contact angle of 30°, above which bilayers degrade or not form at all. Care must be taken as incomplete surface oxidation and hydrophobic recovery result in optically invisible membrane disruption, which will also be transparent to fluorescence microscopy and lipid diffusion measurements in the early stages.

Visualization of Self-Assembly and Hydration of a ß-Hairpin through Integrated Small and Wide-Angle Neutron Scattering.

Fundamental understanding of the structure and assembly of nanoscale building blocks is crucial for the development of novel biomaterials with defined architectures and function. However, accessing self-consistent structural information across multiple length scales is challenging. This limits opportunities to exploit atomic scale interactions to achieve emergent macroscale properties. In this work we present an integrative small- and wide-angle neutron scattering approach coupled with computational modeling to reveal the multiscale structure of hierarchically self-assembled ß hairpins in aqueous solution across 4 orders of magnitude in length scale from 0.1 Å to 300 nm. Our results demonstrate the power of this self-consistent cross-length scale approach and allows us to model both the large-scale self-assembly and small-scale hairpin hydration of the model ß hairpin CLN025. Using this combination of techniques, we map the hydrophobic/hydrophilic character of this model self-assembled biomolecular surface with atomic resolution. These results have important implications for the multiscale investigation of aqueous peptides and proteins, for the prediction of ligand binding and molecular associations for drug design, and for understanding the self-assembly of peptides and proteins for functional biomaterials.

A study of the interaction between TMAO and urea in water using NMR spectroscopy.

Trimethylamine N-oxide (TMAO) and urea are small organic biological molecules. While TMAO is known as a protective osmolyte that promotes the native form of biomolecules, urea is a denaturant. An understanding of the impact of TMAO and urea on water structure may aid in uncovering the molecular mechanisms that underlie this activity. Here we investigate binary solutions of TMAO-water, urea-water and ternary solutions of TMAO-urea-water using NMR spectroscopy at 300 K. An enhancement of the total hydrogen bonding in water was found upon the addition of TMAO and this effect was neutralised by a mole ratio of 1-part TMAO to 4-parts urea. Urea was found to have little effect on the strength of water's hydrogen bonding network and the dynamics of water molecules. Evidence was found for a weak interaction between TMAO and urea. Taken together, these results suggest that TMAO's function as a protective osmolyte, and its counteraction of urea, may be driven by the strength of its hydrogen bond interactions with water, and by a secondary reinforcement of water's own hydrogen bond network. They also suggest that the TMAO-urea complex forms through the donation of a hydrogen bond by urea.

Trimethylamine N-oxide (TMAO) resists the compression of water structure by magnesium perchlorate: terrestrial kosmotrope vs. Martian chaotrope.

The presence of magnesium perchlorate (Mg(ClO4)2) as the dominant ionic compound in the Martian regolith and the recent discovery of a subsurface lake on Mars suggests that beneath the Martian surface may lie an aqueous environment suitable for life, rich in chaotropic ions. Closer to Earth, terrestrial organisms use osmolytes, such as trimethylamine N-oxide (TMAO), to overcome the biologically damaging effects of pressure. While previous studies have revealed that Mg(ClO4)2 acts to modify water structure as if it has been pressurized, little is known about the competing effects of chaotropes and kosmotropes. Here we ask whether TMAO can help to preserve the hydrogen bond network of water against the pressurising effect of Mg(ClO4)2? We address this question using neutron scattering, computational modelling using Empirical Potential Structure Refinement (EPSR) analysis, and a new approach to quantifying hydrogen bond conformations and energies. We find that the addition of 1.0 M TMAO to 0.2 M Mg(ClO4)2 or to 2.7 M Mg(ClO4)2 is capable of partially restoring the hydrogen bond network of water, and the fraction of water molecules in energetically unfavourable conformations. This suggests that terrestrial protecting osmolytes could provide a protective mechanism to the extremes found in Martian environments for biological systems.

The ability of trimethylamine N-oxide to resist pressure induced perturbations to water structure.

Trimethylamine N-oxide (TMAO) protects organisms from the damaging effects of high pressure. At the molecular level both TMAO and pressure perturb water structure but it is not understood how they act in combination. Here, we use neutron scattering coupled with computational modelling to provide atomistic insight into the structure of water under pressure at 4 kbar in the presence and absence of TMAO. The data reveal that TMAO resists pressure-induced perturbation to water structure, particularly in retaining a clear second solvation shell, enhanced hydrogen bonding between water molecules and strong TMAO - water hydrogen bonds. We calculate an 'osmolyte protection' ratio at which pressure and TMAO-induced energy changes effectively cancel out. Remarkably this ratio translates across scales to the organism level, matching the observed concentration dependence of TMAO in the muscle tissue of organisms as a function of depth. Osmolyte protection may therefore offer a molecular mechanism for the macroscale survival of life in extreme environments.

Bridging Structure, Dynamics, and Thermodynamics: An Example Study on Aqueous Potassium Halides.

Aqueous salt systems are ubiquitous in all areas of life. The ions in these solutions impose important structural and dynamic perturbations to water. In this study, we employ a combined neutron scattering, nuclear magnetic resonance, and computational modeling approach to deconstruct ion-specific perturbations to water structure and dynamics and shed light on the molecular origins of bulk thermodynamic properties of the solutions. Our approach uses the atomistic scale resolution offered to us by neutron scattering and computational modeling to investigate how the properties of particular short-ranged microenvironments within aqueous systems can be related to bulk properties of the system. We find that by considering only the water molecules in the first hydration shell of the ions that the enthalpy of hydration can be determined. We also quantify the range over which ions perturb water structure by calculating the average enthalpic interaction between a central halide anion and the surrounding water molecules as a function of distance and find that the favorable anion-water enthalpic interactions only extend to ∼4 Å. We further validate this by showing that ions induce structure in their solvating water molecules by examining the distribution of dipole angles in the first hydration shell of the ions but that this perturbation does not extend into the bulk water. We then use these structural findings to justify mathematical models that allow us to examine perturbations to rotational and diffusive dynamics in the first hydration shell around the potassium halide ions from NMR measurements. This shows that as one moves down the halide series from fluorine to iodine, and ionic charge density is therefore reduced, that the enthalpy of hydration becomes less negative. The first hydration shell also becomes less well structured, and rotational and diffusive motions of the hydrating water molecules are increased. This reduction in structure and increase in dynamics are likely the origin of the previously observed increased entropy of hydration as one moves down the halide series. These results also suggest that simple monovalent potassium halide ions induce mostly local perturbations to water structure and dynamics.

Solute Specific Perturbations to Water Structure and Dynamics in Tertiary Aqueous Solution.

Liquid water is known as the "universal" solvent, capable of dissolving a wide variety of different solutes. While much is now understood about the impact of solutes on the water structure in binary solutions, it is much more challenging to deconvolute the potentially competing effects of more complex solutions. Here, we present a correlative NMR and neutron diffraction study to examine the solute induced perturbation of water structure and dynamics in a tertiary solution containing the naturally occurring osmolyte trimethylamine N-oxide (TMAO) and magnesium perchlorate (Mg(ClO4)2). We show that while TMAO and Mg(ClO4)2 perturb the water structure in an opposing manner, the two solutes slow water dynamics in an additive manner. We quantify the relative ability of each solute to perturb water by introducing a weighting parameter and show that TMAO is 1.54 times more effective at perturbing water structure and dynamics than Mg(ClO4)2. The combination of NMR, neutron diffraction, and computational modelling offers unprecedented access to the structure and dynamics of more complex aqueous solutions, permitting the deconvolution of solute specific perturbation of water. Such insight provides a new route to understand this universal solvent in the context of important and relevant aqueous environments.