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.

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.