Molecular mechanism for the preferential exclusion of TMAO from protein surfaces

Abstract

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.

Figure 1

Osmotic Pressure of TMAO solutions. (A) The schematic illustrates the simulation method described in the text. Water, the primary solvent is shown as a blue background and the cosolvent molecules, shown as orange spheres, are confined to the central region using a restraining potential. (B) The osmotic pressure of TMAO using the new parameters (orange) is shown in contrast to the Kast model (indigo), ideal solution (red) and experimental (cyan) values.

Figure 2

Radial distribution functions in 2 molal TMAO solution. Distributions of TMAO nitrogen (NTM) and water oxygen (OW) around TMAO nitrogen shown for (A) Kast Model and (B) Osmotic Model. Distributions of TMAO oxygen (OTM) and water oxygen (OW) around TMAO oxygen shown for (C) Kast Model and (D) Osmotic Model.

Figure 3

Preferential interaction of TMAO with (A) poly-glycine (B) poly-phenylalanine (C) poly-asparagine (D) poly-aspartic acid. The results for Kast and Osmotic models are shown in black and red respectively.

Figure 4

Thermodynamics of Trpcage stability in presence of TMAO. (A) Temperature dependence of the ellipticity of Trp-cage at different concentrations of TMAO. Solid lines show the results of the fit to a two-state model with the parameters T_m, ΔH(T_m), and ΔG(25°C) shown in (B),(C) and (D) respectively. The solid line in (D) shows the linear regression fit to the data, and the slope of the line corresponds to m-value for TMAO and is estimated to be 0.30 ± 0.15 kJ/(mol M). ±

Figure 5

Preferential interaction of TMAO with Trpcage using (A) Kast model (B) Osmotic model v1 (C) Osmotic-LJ model. The result for the folded and unfolded ensembles are shown in black and red respectively.

Figure 6

Preferential interaction of TMAO with polypetides (A) Polygly (B) Polyphe (C) Polyasn (D) Trpcage (E) Ubiquitin (F) Lysozyme. The Kast model is shown in black, the effect of charge scaling in orange, the effect of reduced epsilon in indigo, and the full Osmotic model in red.

Figure 7

Osmotic pressure and Preferential interaction. (A) Osmotic pressure of 2.5 m TMAO solution as a function of scaling of TMAO charges. α = 1.0 corresponds to the Kast model. (B) Preferential interaction of Trpcage with TMAO as a function of scaling of TMAO charges. (C) Osmotic pressure of 2.5 m urea solution as a function of scaling of urea charges. α = 1.0 corresponds to the KBFF model. (B) Preferential interaction of Trpcage with urea as a function of scaling of TMAO charges.

Figure 8

Osmotic behavior of cosolvents. We hypothesize that osmolytes (orange) show a positive deviation from the van’t Hoff law (black), while denaturants (red) show a negative deviation from the same.