Counteraction of urea-induced protein denaturation by trimethylamine N-oxide: a chemical chaperone at atomic resolution

Abstract

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.

Fig. 1.

Cα RMSD from the crystal structure and the total SASA of CI2 in various solvent environments. (a and c) CI2 in pure water at 60°C and 125°C, in 8 M urea at 60°C, in 1 M TMAO at 25°C, in 4 M TMAO at 60°C, and in a ternary mixture of 8 M urea and 4 M TMAO at 60°C. (b) Simulations at 60°C of 8 M urea and 4 M TMAO/8 M urea, which are displayed also in a.

Fig. 2.

Cα RMSD from the starting structure mapped per residue of CI2 in various solvent environments.

Fig. 3.

The CI2 crystal structure and final 10-ns structures from MD simulations in different solvent environments.

Fig. 5.

Stereoviews of solvent structure in the hydration shell and in bulk solvent. (a) TMAO H bonding with Lys-11 at 10 ns in the 4 M TMAO simulation. (b) Hydration shell of a TMAO molecule in the bulk solvent from the 10-ns snapshot of the 8 M urea/4 M TMAO simulation.

Fig. 4.

H-bond length distributions in bulk solvent for water–water and water–urea H bonds. All data were normalized by the average number of water molecules over the analysis interval (5,000 snapshots) for nonhydration water molecules (>3.5 Å from the protein).

Fig. 6.

The hydration shell around 10-ns structures of CI2 in 8 M urea (a) and 4 M TMAO/8 M urea (b). Green, TMAO; blue, water; red, urea.