Mo(IV) Ion-Modulated BSA-Protected Gold Nanocluster Probe for Fluorescence Turn-On Detection of Trimethylamine N-Oxide (TMAO).

Trimethylamine N-oxide (TMAO), a molecule produced by the microbiota, has been associated with human health and illness. Its early discovery in body fluids may affect our understanding of the pathophysiology and treatment of many illnesses. Therefore, our knowledge of the pathophysiology and diagnostics of disorders associated with TMAO might be enhanced by the creation of dependable and fast methods for TMAO detection. Therefore, we developed a fluorescent probe for detecting TMAO utilizing an on-off-on strategy. Bovine serum albumin (BSA)@AuNCs luminescence is effectively quenched by Mo4+ because BSA@AuNCs and Mo4+ have a strong binding relationship. Mo4+ ions can substantially decrease the emission intensity of gold nanoclusters by establishing a BSA@AuNCs-Mo system. Then, the luminescence of BSA@AuNCs was restored due to the interaction between Mo4+ and TMAO. A significant linear relationship was seen between the emission intensity and TMAO concentration within the 0-201 µM range, with a detection limit of 1.532 µM. Additionally, the method can measure TMAO in blood and urine samples.

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.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Fast real-time monitoring of meat freshness based on fluorescent sensing array and deep learning: From development to deployment.

A fluorescent sensor array (FSA) combined with deep learning (DL) techniques was developed for meat freshness real-time monitoring from development to deployment. The array was made up of copper metal nanoclusters (CuNCs) and fluorescent dyes, having a good ability in the quantitative and qualitative detection of ammonia, dimethylamine, and trimethylamine gases with a low limit of detection (as low as 131.56 ppb) in range of 5 âˆ¼ 1000 ppm and visually monitoring the freshness of various meats stored at 4 °C. Moreover, SqueezeNet was applied to automatically identify the fresh level of meat based on FSA images with high accuracy (98.17 %) and further deployed in various production environments such as personal computers, mobile devices, and websites by using open neural network exchange (ONNX) technique. The entire meat freshness recognition process only takes 5 âˆ¼ 7 s. Furthermore, gradient-weighted class activation mapping (Grad-CAM) and uniform manifold approximation and projection (UMAP) explanatory algorithms were used to improve the interpretability and transparency of SqueezeNet. Thus, this study shows a new idea for FSA assisted with DL in meat freshness intelligent monitoring from development to deployment.

Structurally different chemical chaperones show similar mechanical roles with independent molecular mechanisms.

Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are mechanistically complicated and context dependent, the underlying molecular mechanism is not fully understood. Here, we combined single-molecule magnetic tweezers and molecular dynamics (MD) simulation to explore the mechanical role of osmolytes from two different classes, trimethylamine N-oxide (TMAO) and trehalose, as mechanical stabilizers of protein structure. We observed that these osmolytes increase the protein L mechanical stability by decreasing unfolding kinetics while accelerating the refolding kinetics under force, eventually shifting the energy landscape toward the folded state. These osmolytes mechanically stabilize the protein L and plausibly guide them to more thermodynamically robust states. Finally, we observed that osmolyte-modulated protein folding increases mechanical work output up to twofold, allowing the protein to fold under a higher force regime and providing a significant implication for folding-induced structural stability in proteins.

Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress.

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

Molecular Aggregation Behavior and Microscopic Heterogeneity in Binary Osmolyte-Water Solutions.

Osmolytes, small organic compounds, play a key role in modulating the protein stability in aqueous solutions, but the operating mechanism of the osmolyte remains inconclusive. Here, we attempt to clarify the mode of osmolyte action by quantitatively estimating the microheterogeneity of osmolyte-water mixtures with the aid of molecular dynamics simulation, graph theoretical analysis, and spatial distribution measurement in the four osmolyte solutions of trimethylamine-N-oxide (TMAO), tetramethylurea (TMU), dimethyl sulfoxide, and urea. TMAO, acting as a protecting osmolyte, tends to remain isolated with no formation of osmolyte aggregates while preferentially interacting with water, but there is a strong aggregation propensity in the denaturant TMU solution, characterized by favored hydrophobic interactions between TMU molecules. Taken together, the mechanism of osmolyte action on protein stability is proposed as a comprehensive one that encompasses the direct interactions between osmolytes and proteins and indirect interactions through the regulation of water properties in the osmolyte-water mixtures.

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.

TMAO Destabilizes RNA Secondary Structure via Direct Hydrogen Bond Interactions.

Trimethylamine N-oxide (TMAO) is an osmolyte that accumulates in cells in response to osmotic stress. TMAO stabilizes proteins by the entropic stabilization mechanism, which pictures TMAO as a nanocrowder that predominantly destabilizes the unfolded state. However, the mechanism of action of TMAO on RNA is much less understood. Here, we use all-atom molecular dynamics simulations to investigate how TMAO interacts with a 12-nt RNA hairpin with a high melting temperature, and an 8-nt RNA hairpin, which has a relatively fluid native basin in the absence of TMAO. The use of the two hairpins with different free energy of stabilization allows us to probe the origin of the destabilization effect of TMAO on RNA molecules without the possibility of forming tertiary interactions. We generated multiple trajectories using all-atom molecular dynamics (MD) simulations in explicit water by employing AMBER and CHARMM force fields, both in the absence and presence of TMAO. We observed qualitatively similar RNA-TMAO interaction profiles from the simulations using the two force fields. TMAO hydrogen bond interactions are largely depleted around the paired RNA bases and ribose sugars. In contrast, we show that the oxygen atom in TMAO, the hydrogen bond acceptor, preferentially interacts with the hydrogen bond donors in the solvent exposed bases, such as those in the stem-loop and the destabilized base stacks in the unfolded state, especially in the marginally stable 8-nt RNA hairpin. The predicted destabilization mechanism through TMAO-RNA hydrogen bond interactions could be tested using two-dimensional IR spectroscopy. Since TMAO does not significantly interact with the hydroxyl group of the ribose sugars, we predict that similar results must also hold for DNA.

Urea counteracts trimethylamine N-oxide (TMAO) compaction of lipid membranes by modifying van der Waals interactions.

To cope with stress induced by high salinity and hydrostatic pressure, some marine animals accumulate small organic solutes called osmolytes. Most notable among these osmolytes are the denaturant urea, and trimethylamine N-oxide (TMAO) that is known to stabilize proteins. Although their effects on proteins and nucleic acids have been extensively studied, osmolytes are less commonly studied in the context of lipids, which are a crucial component in many cellular processes. Here we resolve the mechanism for urea's action on the forces acting between lipid membranes, in the presence and absence of TMAO. We find that unlike the way urea denatures proteins, and by contrast to TMAO, urea does not preferentially interact with net-neutral lipid membranes. Instead, urea modulates the interactions between membranes mainly by weakening the van der Waals attraction between bilayers. Interestingly, regardless of concentration, the effects of urea and TMAO appear to be additive to a large extent, so that the presence of one osmolyte hardly changes the interaction of the other with lipid. Weak non-additive effects are likely due to structural changes in the lipid membrane induced by the osmolytes. Finally, we comment on the potential role of osmolytes acting together in the modification of lipid adhesion and fusion.

Osmolyte effect on enzymatic stability and reaction equilibrium of formate dehydrogenase.

Osmolytes are well-known biocatalyst stabilisers as they promote the folded state of proteins, and a stabilised biocatalyst might also improve reaction kinetics. In this work, the influence of four osmolytes (betaine, glycerol, trehalose, and trimethylamine N-oxide) on the activity and stability of Candida bondinii formate dehydrogenase cbFDH was studied experimentally and theoretically. Scanning differential fluorimetric studies were performed to assess the thermal stability of cbFDH, while UV detection was used to reveal changes in cbFDH activity and reaction equilibrium at osmolyte concentrations between 0.25 and 1 mol kg-1. The thermodynamic model ePC-SAFT advanced allowed predicting the effects of osmolyte on the reaction equilibrium by accounting for interactions involving osmolyte, products, substrates, and water. The results show that osmolytes at low concentrations were beneficial for both, thermal stability and cbFDH activity, while keeping the equilibrium yield at high level. Molecular dynamics simulations were used to describe the solvation around the cbFDH surface and the volume exclusion effect, proofing the beneficial effect of the osmolytes on cbFDH activity, especially at low concentrations of trimethylamine N-oxide and betaine. Different mechanisms of stabilisation (dependent on the osmolyte) show the importance of studying solvent-protein dynamics towards the design of optimised biocatalytic processes.

Ultrasensitive Flexible Olfactory Receptor-Derived Peptide Sensor for Trimethylamine Detection by the Bending Connection Method.

Trimethylamine (TMA) is a harmful gas that exists ubiquitously in the environment; therefore, the sensitive and specific monitoring of TMA is necessary. In this work, we prepared ultrasensitive flexible sensors for TMA detection based on single-walled carbon nanotubes (SWCNTs) and olfactory receptor-derived peptides (ORPs) on low-cost plastic substrates. A novel bending connection method was developed by intentionally bending the interdigitated electrodes with SWCNTs to form a three-dimensional structure during the ORP-connection process, leading to the exposure of more modification sites. The method showed ∼4.7-fold more effective connection amount of the ORPs to SWCNTs compared to the conventional flat-condition connection method. The flexible ORP-SWCNT sensors could significantly improve the limit of detection for gaseous TMA from the reported lowest limit of 10 parts per quadrillion (ppq) to 0.1 ppq. The flexible ORP sensors also exhibited excellent sensitivity to vaporized TMA standards and TMA generated by different kinds of foods under different bending conditions. The results showed that the bending connection method in this work was effective for ultrasensitive flexible ORP sensors and their associated applications.

Electronic fluctuation difference between trimethylamine N-oxide and tert-butyl alcohol in water.

Although small organic molecules in cells have been considered important to control the functions of proteins, their electronic fluctuation and the intermolecular interaction, which is physicochemical origin of the molecular functions, under physiological conditions, i.e., dilute aqueous solutions (0.18 mol L-1), has never been clarified due to the lack of observation methods with both accuracy and efficiency. Herein, the time evolutions of the interactions in dilute aqueous trimethylamine N-oxide (TMAO) and tert-butyl alcohol (TBA) solutions were analyzed via ab initio molecular dynamics simulations accelerated with the fragment molecular theory. It has been known that TMAO and TBA have similar structures, but opposite physiological functions to stabilize and destabilize proteins. It was clarified that TMAO induced stable polarization and charge-transfer interactions with water molecules near the hydrophilic group, and water molecules were caught even near the CH3- group. Those should affect protein stabilization. Understanding the solution dynamics will contribute to artificial chaperone design in next generation medicine.

Influence of TMAO and Pressure on the Folding Equilibrium of TrpCage.

Trimethylamine-N-oxide (TMAO) is an osmolyte known for its ability to counteract the pressure denaturation of proteins. Computational studies addressing the molecular mechanisms of TMAO's osmolyte action have however focused exclusively on its protein-stabilizing properties at ambient pressure, neglecting the changes that may occur under high-pressure conditions where TMAO's hydration structure changes to that of increased water binding. Here, we present the first study on the combined effect of pressure and TMAO on a mini-protein, TrpCage. The results showed that at high pressures, nonpolar residues packed less tightly and the salt bridge of TrpCage was destabilized. This effect was mitigated by TMAO which was found to be strongly depleted from the protein/water interface at 1 kbar than at 1 bar ambient pressure, thus counterbalancing the thermodynamically unfavorable effect of elevated pressure in the free energy of folding. TMAO was depleted from charged groups, like the salt bridge-forming ones, and accumulated around hydrophobic groups. Still, it stabilized both kinds of interactions. Furthermore, enthalpically favorable TrpCage-water hydrogen bonds were reduced in the presence of TMAO, causing a stronger destabilization of the unfolded state than the folded state. This shifted the protein-folding equilibrium toward the folded state. Therefore, TMAO showed stabilizing effects on different kinds of groups, which were partially enhanced at high pressures.

The protein-stabilizing effects of TMAO in aqueous and non-aqueous conditions.

We present a new water-dependent molecular mechanism for the widely-used protein stabilizing osmolyte, trimethylamine N-oxide (TMAO), whose mode of action has remained controversial. Classical interpretations, such as osmolyte exclusion from the vicinity of protein, cannot adequately explain the behavior of this osmolyte and were challenged by recent data showing the direct interactions of TMAO with proteins, mainly via hydrophobic binding. Solvent effect theories also fail to propose a straightforward mechanism. To explore the role of water and the hydrophobic association, we disabled osmolyte-protein hydrophobic interactions by replacing water with hexane and using lipase enzyme as an anhydrous-stable protein. Biocatalysis experiments showed that under this non-aqueous condition, TMAO does not act as a stabilizer, but strongly deactivates the enzyme. Molecular dynamics (MD) simulations reveal that TMAO accumulates near the enzyme and makes many hydrogen bonds with it, like denaturing osmolytes. Some TMAO molecules even reach the active site and interact strongly with the catalystic traid. In aqueous solvent, the enzyme functions well: the extent of TMAO interactions is reduced and can be divided into both polar and non-polar terms. Structural analysis shows that in water, some TMAO molecules bind to the enzyme surface like a surfactant. We show that these interactions limit water-protein hydrogen bonds and unfavorable water-hydrophobic surface contacts. Moreover, a more hydrophobic environment is formed in the solvation layer, which reduces water dynamics and subsequently, rigidifies the backbone in aqueous solution. We show that osmolyte amphiphilicity and protein surface heterogeneity can address the weaknesses of exclusion and solvent effect theories about the TMAO mechanism.

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.

Cosolvent Exclusion Drives Protein Stability in Trimethylamine N-Oxide and Betaine Solutions.

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein-osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood-Buff integrals and density. We further introduced appropriate scaling to protein-osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein-osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein-TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.

Kinetics, Products, and Brown Carbon Formation by Aqueous-Phase Reactions of Glycolaldehyde with Atmospheric Amines and Ammonium Sulfate.

Glycolaldehyde (GAld) is a C2 water-soluble aldehyde produced during the atmospheric oxidation of isoprene and many other species and is commonly found in cloudwater. Previous work has established that glycolaldehyde evaporates more readily from drying aerosol droplets containing ammonium sulfate (AS) than does glyoxal, methylglyoxal, or hydroxyacetone, which implies that it does not oligomerize as quickly as these other species. Here, we report NMR measurements of glycolaldehyde's aqueous-phase reactions with AS, methylamine, and glycine. Reaction rate constants are smaller than those of respective glyoxal and methylglyoxal reactions in the pH range of 3-6. In follow-up cloud chamber experiments, deliquesced glycine and AS seed particles were found to take up glycolaldehyde and methylamine and form brown carbon. At very high relative humidity, these changes were more than 2 orders of magnitude faster than predicted by our bulk liquid NMR kinetics measurements, suggesting that reactions involving surface-active species at crowded air-water interfaces may play an important role. The high-resolution liquid chromatography-electrospray ionization-mass spectrometric analysis of filter extracts of unprocessed AS + GAld seed particles identified sugar-like C6 and C12 GAld oligomers, including proposed product 3-deoxyglucosone, with and without modification by reactions with ammonia to diimine and imidazole forms. Chamber exposure to methylamine gas, cloud processing, and simulated sunlight increased the incorporation of both ammonia and methylamine into oligomers. Many C4-C16 imidazole derivatives were detected in an extract of chamber-exposed aerosol along with a predominance of N-derivatized C6 and C12 glycolaldehyde oligomers, suggesting that GAld is capable of forming brown carbon SOA.

Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress.

Because organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine-N-oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein.

Temperature induced change of TMAO effects on hydrophobic hydration.

The effect of trimethylamine-N-oxide (TMAO) on hydrophobic solvation and hydrophobic interactions of methane has been studied with Molecular Dynamics simulations in the temperature range between 280 and 370 K at 1 bar ambient pressure. We observe a temperature transition in the effect of TMAO on the aqueous solubility of methane. At low temperature (280 K), methane is preferentially hydrated, causing TMAO to reduce its solubility in water, while above 320 K, methane preferentially interacts with TMAO, causing TMAO to promote its solubility in water. Based on a statistical-mechanical analysis of the excess chemical potential of methane, we find that the reversible work of creating a repulsive methane cavity opposes the solubility of methane in TMAO/water solution more than in pure water. Below 320 K, this solvent-excluded volume effect overcompensates the contribution of methane-TMAO van der Waals interactions, which promote the solvation of methane and are observed at all temperatures. These van der Waals interactions with the methyl groups of TMAO tip the balance above 320 K where the effect of TMAO on solvent-excluded volume is smaller. We furthermore find that the effective attraction between dissolved methane solutes increases with the increasing TMAO concentration. This observation correlates with a reduction in the methane solubility below 320 K but with an increase in methane solubility at higher temperatures.