trimethylamine-N-oxide Molecules Research Articles

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.

TMAO perturbs intermolecular vibrational motions of water revealed by low-frequency modes.

Trimethylamine N-oxide (TMAO) as a representative natural osmolyte has received much attention because of its unique properties, including enhancement of hydrogen bonding networks in liquid water and stabilization of three-dimensional structures of proteins in living organisms. As a hydrogen bond maker and/or a protein stabilizer, its hydrated structures and orientation dynamics in aqueous solutions have been investigated by various spectroscopic methods. Particularly, distinct from other natural osmolytes, it has been found that TMAO molecules form complexes with water molecules even at low concentrations, showing extraordinarily long lifetimes and much larger effective dipole moments. In this study, we demonstrated that collective motions of water molecules are closely correlated to TMAO molecules, as revealed by the changes of the librational modes observed in hyper-Raman (HR) spectra in the low-frequency region (<1000 cm-1) for the first time. Based on HR spectra of the TMAO solutions at submolar concentrations, we observed that the librational bands originating from water apparently upshift (∼15 cm-1) upon the addition of TMAO molecules. Compared to the OH stretching band of water showing a negligible downshift (<5 cm-1), the librational bands of water are more sensitive to reflect changes in the hydrogen bonding networks in the TMAO solutions, suggesting formation of transient TMAO-water complexes plays an essential role toward surrounding water molecules in perturbing their librational motions. We expect to provide a supplementary approach to understand that water molecules in TMAO aqueous solutions are strongly affected by TMAO molecules, different from other osmolytes.

Counteracting Effects of Trimethylamine N-Oxide against Urea in Aqueous Solutions: Insights from Theoretical Two-Dimensional Infrared Spectroscopy.

The study of small osmolytes in their aqueous solutions has gained significant attention because of their relevance to structure and thermodynamics of proteins in aqueous media. Special attention has been given to the binary and ternary aqueous solutions of urea and trimethylamine N-oxide (TMAO). Urea is a well-known protein denaturant, while TMAO protects proteins in their native states. Interestingly, TMAO counteracts urea's ability to denature proteins when present in solutions with approximately half of the concentration of urea. Vibrational spectroscopy can improve our understanding of the molecular origin of this counteracting effect because of its sensitivity to local structure and dynamics. We present results of theoretical linear vibrational and two-dimensional infrared (2DIR) spectroscopy of water in the binary and ternary aqueous solutions of TMAO and urea. The 2DIR spectra are calculated using the electronic structure/molecular dynamics approach. The non-Condon effects in spectral transitions are incorporated in the theoretical calculations of 2DIR spectra. It is found that TMAO disrupts the local structure of water, while urea leaves it essentially unaffected. The 2DIR results show that both TMAO and urea slow down the dynamics of spectral diffusion of water. The extent of slowing down is found to be particularly significant for both hydration and bulk water in the presence of TMAO which can be attributed to strong hydrogen bonds between the water and TMAO molecules. The water molecules present in the hydration layer of the solutes in the ternary solutions are found to relax at even slower rates compared to that in their binary solutions in water. The hydrogen bonds between TMAO and urea are found to be not stable. Thus, the counteracting effect of TMAO against urea is seen to take place mainly through water-mediated interactions. Such TMAO-induced effects giving rise to more structured and slower hydrogen-bonded network are successfully captured through 2DIR spectroscopic calculations.

TMAO: Protecting proteins from feeling the heat

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 μs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules “herding” water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Raman spectroscopic, computational, and X-ray crystallographic investigation of intermolecular interactions in trimethylamine N-oxide (TMAO) and TMAO-d9

Crystalline structures of trimethylamine N-oxide (TMAO) and its isomorphic, perdeuterated analogue (TMAO-d9) were investigated using Raman spectroscopy, X-ray crystallography, and electronic structure theory. Shifts to higher vibrational energy were observed in both Raman Under liquid Nitrogen Spectroscopy (RUNS) and high pressure Raman spectroscopy studies. Agreement between experimental Raman spectra and electronic structure computations performed on individual TMAO molecules was poor; however, better agreement was obtained when model unit cells were considered. These results also show that TMAO-d9′s spectra are significantly less perturbed than those for TMAO, suggesting weaker interactions between neighboring TMAO molecules when deuterated.

Effect of TMAO on the Structure and Phase Transition of Lipid Membranes: Potential Role of TMAO in Stabilizing Cell Membranes under Osmotic Stress.

Extremophiles adopt strategies to deal with different environmental stresses, some of which are severely damaging to their cell membrane. To combat high osmotic stress, deep-sea organisms synthesize osmolytes, small polar organic molecules, like trimethylamine-N-oxide (TMAO), and incorporate them in the cell. TMAO is known to protect cells from high osmotic or hydrostatic pressure. Several experimental and simulation studies have revealed the roles of such osmolytes on stabilizing proteins. In contrast, the effect of osmolytes on the lipid membrane is poorly understood and broadly debated. A recent experiment has found strong evidence of the possible role of TMAO in stabilizing lipid membranes. Using the molecular dynamics (MD) simulation technique, we have demonstrated the effect of TMAO on two saturated fully hydrated lipid membranes in their fluid and gel phases. We have captured the impact of TMAO's concentration on the membrane's structural properties along with the fluid/gel phase transition temperatures. On increasing the concentration of TMAO, we see a substantial increase in the packing density of the membrane (estimated by area, thickness, and volume) and enhancement in the orientational order of lipid molecules. Having repulsive interaction with the lipid head group, the TMAO molecules are expelled away from the membrane surface, which induces dehydration of the lipid head groups, increasing the packing density. The addition of TMAO also increases the fluid/gel phase transition temperature of the membrane. All of these results are in close agreement with the experimental observations. This study, therefore, provides a molecular-level understanding of how TMAO can influence the cell membrane of deep-sea organisms and help in combating the osmotic stress condition.

Statistical geometry characterization of local structure of TMAO, TBA and urea aqueous solutions

Recently, we have revealed that trimethylamine-N-oxide (TMAO) molecules in aqueous solutions are distributed generally like random spheres in space (A.V. Anikeenko et al. J. Mol. Liq. 245 (2017) 35–41). We have shown that the variance of the distribution of Voronoi region volumes for the TMAO molecules and the hard random spheres are identical at the corresponding concentrations up to the mole fraction x ~ 0.1 (or packing fraction η ~ 0.2). In the current work, we study clusters in TMAO solutions and calculate the weighted mean cluster size and some topological cluster characteristics. For all these parameters, a great matching of TMAO and random spheres is also observed. The same analysis for tert-butyl alcohol (TBA) and urea aqueous solutions is carried out. These molecules have a tendency to cluster even at very low concentrations. However, basic geometrical features of the clusters are the same both for solutions and for the system of random spheres, indicating that geometrical laws governing the allocation of non-overlapping spheres in space should be taken into account when describing the structure of solutions even at rather low concentrations.

Подвижность молекул воды, мочевины и триметиламиноксида вблизи глобулярного белка

C использованием полноатомного молекулярно-динамического моделирования исследовали подвижность молекул в гидратной оболочке глобулярного белка SNase в водном растворе с косольвентами (мочевина и триметиламиноксид). Для молекул, изначально расположенных в последовательных слоях вокруг белка, рассчитывали средние смещения за один и тот же короткий промежуток времени. Это позволило характеризовать диффузионную подвижность молекул в зависимости от расстояния до белка. Показано, что молекулы растворителя имеют пониженную подвижность вблизи белка, а при удалении от него подвижность молекул увеличивается неравномерно, коррелируя с положением максимумов функции распределения соответствующих молекул вокруг белка. За пределами второго максимума этих функций подвижность достигает своих объемных значений как для воды, так и для косольвентов.

Atomistic level understanding of the stabilization of protein Trp cage in denaturing and mixed osmolyte solutions

Using replica exchange molecular dynamics simulation technique we report urea-induced denaturation and its counteraction by TMAO (Trimethylamine-N-oxide) of protein Trp cage. We find that in binary solution urea molecules are very much prone to stay nearby the salt bridge. In presence of TMAO, salt bridge is protected from urea molecules. TMAO also prevents hydration of the hydrophobic core of the protein and thus provides counteraction. Emphasizing on the orientational preferences of different aromatic planes reveals that near parallel orientation of Trp6-Trp18 and near perpendicular orientations of Trp6-Trp17 and Trp6-Trp19 have significant contributions towards stabilization. Trp6-Tyr3 pair is more likely to maintain a tilt angle of 25°. These orientational preferences of the planes that are lost in binary urea solution regained significantly in mixed urea/TMAO solutions. We have also found a highly favorable interaction energy between protein and urea contributed from both electrostatic and van der Waals components. Addition of TMAO makes these interactions more unfavorable. Protein solvation behavior and hydrogen bond calculations show that TMAO molecules replace some urea molecules from the surface of the protein. As a result, a reduction in the protein-urea hydrogen bonds is observed when compared to that of binary urea system.

Statistical geometry characterization of global structure of TMAO and TBA aqueous solutions

We study the spatial distribution of trimethylamine-N-oxide (TMAO) and tert-butyl alcohol (TBA) molecules in aqueous solutions at low concentrations using molecular dynamics simulation, Voronoi-Delaunay method, and statistical cluster analysis. A comparison of these solutions with the systems of randomly distributed hard spheres is carried out. It is shown that TMAO molecules are generally distributed like random spheres. On the contrary, the distribution of TBA substantially differs from the random one, being the result of the self-association process in TBA solutions. Thus, using the methods of statistical geometry and systems of random spheres as a reference system, one can carry out a quantitative characterization of a global structure of a solution to describe general features of the spatial distribution of the solute molecules.

TMAO and urea in the hydration shell of the protein SNase.

We performed all-atom MD simulations of the protein SNase in aqueous solution and in the presence of two major osmolytes, trimethylamine-N-oxide (TMAO) and urea, as cosolvents at various concentrations and compositions and at different pressures and temperatures. The distributions of the cosolvent molecules and their orientation in the surroundings of the protein were analyzed in great detail. The distribution of urea is largely conserved near the protein. It varies little with pressure and temperature, and does practically not depend on the addition of TMAO. The slight decrease with temperature of the number of urea molecules that are in contact with the SNase molecule is consistent with the view that the interaction of the protein with urea is mainly of enthalpic nature. Most of the TMAO molecules tend to be oriented to the protein by its methyl groups, a small amount of these molecules contact the protein by its oxygen, forming hydrogen bonds with the protein, only. Unlike urea, the fraction of TMAO in the hydration shell of SNase slightly increases with temperature (a signature of a prevailing hydrophobic interaction between TMAO and SNase), and decreases significantly upon the addition of urea. This behavior reflects the diverse nature of the interaction of the two osmolytes with the protein. Using the Voronoi volume of the atoms of the solvent molecules (water, urea, TMAO), we compared the fraction of the volume occupied by a given type of solvent molecule in the hydration shell and in the bulk solvent. The volume fraction of urea in the hydration shell is more than two times larger than in the bulk, whereas the volume fraction of TMAO in the hydration shell is only slightly larger in the binary solvent (TMAO + water) and becomes even less than in the bulk in the ternary solvent (TMAO + water + urea). Thus, TMAO tends to be excluded from the hydration shell of the protein. The behavior of the two cosolvents in the vicinity of the protein does not change much with pressure (from 1 to 5000 bar) and temperature (from 280 to 330 K). This is also in line with the conception of the "osmophobic effect" of TMAO to protect proteins from denaturation also at harsh environmental conditions. We also calculated the volumetric parameters of SNase and found that the cosolvents have a small but significant effect on the apparent volume and its contributions, i.e. the intrinsic, molecular and thermal volumes.

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.

Can trimethylamine-N-oxide act to influence the self-aggregation of tert-butyl alcohol?

ABSTRACTWe report classical molecular dynamics simulation studies of aqueous solution consisting of water, tert-butyl alcohol (TBA) and trimethylamine-N-oxide (TMAO). In spite of the fact that both TBA and TMAO molecules have very similar geometry with hydrophobic and hydrophilic groups, they behave very differently in aqueous solutions. Our aim is to see the role of TMAO on the self-aggregation (or association) of TBA molecules. We observe that, TMAO acts to postpone the aggregation of TBA molecules (takes place via the hydrophobic ends) to some extent. Addition of 0.10 mole fraction of TMAO shifts the aggregation concentration of TBA from xtba = 0.025 to xtba = 0.06. From the excess coordination number, calculation it is noticed that up to xtba = 0.06, TBA molecules are favourably solvated by TMAO by replacing water molecules from TBA solvation shell but above this concentration, TBA–TMAO interaction decreases. This is further confirmed by water–TMAO interactions which shows a shift above xtba = 0.06 indicating more preferred interactions between them. We also observe a noticeable increase in the water–water hydrogen bond life time in presence of TBA molecules indicating more structuring of water molecules.

Model Dependency of TMAO’s Counteracting Effect Against Action of Urea: Kast Model versus Osmotic Model of TMAO

Classical molecular dynamics simulation of GB1 peptide (a 16-residue β-hairpin) in different osmotic environments is studied. Urea is used for denaturation of the peptide, and trimethylamine-N-oxide (TMAO) is used to offset the effect of urea. Protein-urea electrostatic interactions are found to play a major role in protein-denaturation. To emphasize on protein protecting action of TMAO against urea, two different models of TMAO are used, viz., the Kast model and the Osmotic model. We observe that the Osmotic model of TMAO gives the best protection to counteract urea's action when used in ratio 1:2 of urea:TMAO (i.e., reverse ratio). This is because the presence of TMAO makes urea-protein electrostatic interactions more unfavorable. Preferential solvation of TMAO molecules by urea (and water) molecules is also observed, which causes depletion in the number of urea molecules in the vicinity of the protein. The calculations of intraprotein hydrogen bonds between different residues of protein further reveal the breaking of backbone hydrogen bonds of residues 2 and 15 in the presence of urea, and the same is preserved in the presence of TMAO. Free energy landscapes show that the narrowest distribution is obtained for the osmotic TMAO model when used in reverse ratio.

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford-Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard-Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system.

Interactions of S-peptide analogue in aqueous urea and trimethylamine-N-oxide solutions: A molecular dynamics simulation study

The ability of the osmolyte, trimethylamine-N-oxide (TMAO), to protect proteins from deleterious effect of urea, another commonly available osmolyte, is well-established. However, the molecular mechanism of this counteraction is not understood yet. To provide a molecular level understanding of how TMAO protects proteins in highly concentrated urea solution, we report here molecular dynamics simulation results of a 15-residue model peptide in two different conformations: helix and extended. For both conformations, simulations are carried out in pure water as well as in binary and ternary aqueous solutions of urea and TMAO. Analysis of solvation characteristics reveals direct interactions of urea and TMAO with peptide residues. However, the number of TMAO molecules that enter in the first solvation shell of the peptide is significantly lower than that of urea, and, unlike water and urea, TMAO shows its inability to form hydrogen bond with backbone oxygen and negatively charged sidechains. Preferential accumulation of urea near the peptide surface and preferential exclusion of TMAO from the peptide surface are observed. Inclusion of osmolytes in the peptide solvation shell leads to dehydration of the peptide in binary and ternary solutions of urea and TMAO. Solvation of peptide residues are investigated more closely by calculating the number of hydrogen bonds between the peptide and solution species. It is found that number of hydrogen bonds formed by the peptide with solution species increases in binary urea solution (relative to pure water) and this relative enhancement in hydrogen bond number reduces upon addition of TMAO. Our simulation results also suggest that, in the ternary solution, the peptide solvation layer is better mixed in terms of water and urea as compared to binary urea solution. Implications of the results for counteraction mechanism of TMAO are discussed.

Molecular Mechanism for the Preferential Exclusion of Osmolytes from Protein Surfaces

Trimethylamine N-oxide (TMAO) is a naturally occurring protecting osmolyte that stabilizes the folded state of proteins and also counteracts the destabilizing effect of urea on protein stability. Experimentally, it has been inferred that TMAO is preferentially excluded from the vicinity of protein surfaces. Here, we combine computer modeling and experimental measurements to gain an understanding of the mechanism of the protecting effect of TMAO on proteins. We have developed an all-atom molecular model for TMAO that captures the exclusion of TMAO from model compounds and protein surfaces, as a consequence of incorporating realistic TMAO-water interactions through osmotic pressure measurements. Osmotic pressure measurements also suggest no significant attraction between urea and TMAO molecules in solution. To obtain an accurate potential for molecular simulations of protein stability in TMAO solutions, we have explored different ways of parameterizing the protein/osmolyte and osmolyte/osmolyte interactions by scaling charges and the strength of Lennard-Jones interactions and carried out equilibrium folding experiments of Trp-cage miniprotein in presence of TMAO to guide the parameterization. Our calculations suggest a general principle for preferential interaction behavior of cosolvents with protein surfaces - preferentially excluded osmolytes have repulsive self-interaction given by osmotic coefficient larger than one, while denaturants, in addition to having attractive interactions with the proteins, have favorable self-interaction given by osmotic coefficient less than one to enable preferential accumulation in the vicinity of proteins. This work has been funded by the National Science Foundation (NSF MCB-1050966 to A.E.G. and MCB-0110396 to G.I.M.) and the National Institutes of Health (GM086801). This research was partially supported by the Intramural Research Program of the National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development.

Molecular Mechanism for the Preferential Exclusion of TMAO from Protein Surfaces

Trimethylamine N-oxide (TMAO) is a naturally occurring protecting osmolyte that stabilizes the folded state of proteins and also counteracts the destabilizing effect of urea on protein stability. Experimentally, it has been inferred that TMAO is preferentially excluded from the vicinity of protein surfaces. Here, we combine computer modeling and experimental measurements to gain an understanding of the mechanism of the protecting effect of TMAO on proteins. We have developed an all-atom molecular model for TMAO that captures the exclusion of TMAO from model compounds and protein surfaces, as a consequence of incorporating realistic TMAO-water interactions through osmotic pressure measurements. Osmotic pressure measurements also suggest no significant attraction between urea and TMAO molecules in solution. To obtain an accurate potential for molecular simulations of protein stability in TMAO solutions, we have explored different ways of parametrizing the protein/osmolyte and osmolyte/osmolyte interactions by scaling charges and the strength of Lennard-Jones interactions and carried out equilibrium folding experiments of Trp-cage miniprotein in the presence of TMAO to guide the parametrization. Our calculations suggest a general principle for preferential interaction behavior of cosolvents with protein surfaces--preferentially excluded osmolytes have repulsive self-interaction given by osmotic coefficient φ > 1, while denaturants, in addition to having attractive interactions with the proteins, have favorable self-interaction given by osmotic coefficient φ < 1, to enable preferential accumulation in the vicinity of proteins.

The effect of aqueous solutions of trimethylamine-N-oxide on pressure induced modifications of hydrophobic interactions

To understand the mechanism of protein protection by the osmolyte trimethylamine-N-oxide (TMAO) at high pressure, using molecular dynamics (MD) simulations, solvation of hydrophobic group is probed in aqueous solutions of TMAO over a wide range of pressures relevant to protein denaturation. The hydrophobic solute considered in this study is neopentane which is a considerably large molecule. The concentrations of TMAO range from 0 to 4 M and for each TMAO concentration, simulations are performed at five different pressures ranging from 1 atm to 8000 atm. Potentials of mean force are calculated and the relative stability of solvent-separated state over the associated state of hydrophobic solute are estimated. Results suggest that high pressure reduces association of hydrophobic solutes. From computations of site-site radial distribution function followed by analysis of coordination number, it is found that water molecules are tightly packed around the nonpolar particle at high pressure and the hydration number increases with increasing pressure. On the other hand, neopentane interacts preferentially with TMAO over water and although hydration of neopentane reduces in presence of this osmolyte, TMAO does not show any tendency to prevent the pressure-induced dispersion of neopentane moieties. It is also observed that TMAO molecules prefer a side-on orientation near the neopentane surface, allowing its oxygen atom to form favorable hydrogen bonds with water while maintaining some hydrophobic contacts with neopentane. Analysis of hydrogen-bond properties and solvation characteristics of TMAO reveals that TMAO can form hydrogen bonds with water and it reduces the identical nearest neighbor water molecules caused by high hydrostatic pressures. Moreover, TMAO enhances life-time of water-water hydrogen bonds and makes these hydrogen bonds more attractive. Implication of these results for counteracting effect of TMAO against protein denaturation at high pressures are discussed.

Complex Formation in Aqueous Trimethylamine-N-oxide (TMAO) Solutions

We study aqueous solutions of the amphiphilic osmolyte trimethylamine-N-oxide (TMAO) using broadband dielectric spectroscopy and femtosecond mid-infrared spectroscopy. Both experiments provide strong evidence for distinctively slower rotation dynamics for water molecules interacting with the hydrophobic part of the TMAO molecules. Further, water is found to interact more strongly at the hydrophilic site of the TMAO molecules: we find evidence for the formation of stable, TMAO·2H2O and/or TMAO·3H2O complexes. While this coordination structure seems obvious, the lifetime of these complexes is found to be extraordinarily long (>50 ps). The existence of these long-lived complexes leads to pronounced parallel dipole correlations between water and TMAO, reflected in enhanced amplitudes in the dielectric spectra. The strong interaction between water and TMAO also results in a red-shifted band for the O-D stretching vibration of HDO molecules in an isotopically diluted aqueous TMAO solution. This O-D stretching vibration has a vibrational lifetime of 670 fs, which is significantly shorter than the lifetime of the O-D stretch vibration of bulk-like HDO molecules, presumably due to efficient coupling to vibrational modes of TMAO. The rotational dynamics of these O-D groups are slowed down dramatically, and are limited by the rotation of the whole complex, while the O-D vector oriented away from TMAO probably shows an accelerated reorientation.