Synergy in protein-osmolyte mixtures

Abstract

Virtually all taxa use osmolytes to protect cells against biochemical stress. Osmolytes often occur in mixtures, such as the classical combination of urea with TMAO (trimethylamine N-oxide) in cartilaginous fish or the cocktail of at least six different osmolytes in the kidney. The concentration patterns of osmolyte mixtures found in vivo make it likely that synergy between them plays an important role. Using statistical mechanical n-component Kirkwood-Buff theory, we show from first principles that synergy in protein-osmolyte systems can arise from two separable sources: (1) mutual alteration of protein surface solvation and (2) effects mediated through bulk osmolyte chemical activities. We illustrate both effects in a four-component system with the experimental example of the unfolding of a notch ankyrin domain in urea-TMAO mixtures, which make urea a less effective denaturant and TMAO a more effective stabilizer. Protein surface effects are primarily responsible for this synergy. The specific patterns of surface solvation point to denatured state expansion as the main factor, as opposed to direct competition.

Figure 1

Effect of urea–TMAO mixtures on three proteins. Urea-induced unfolding at variable TMAO concentrations of Nank4–7*, Nank1–7*, and barnase (A–C). Molar TMAO concentrations (from left to right) are 0, 1/8, 1/4, 3/8, 1/2, and 3/4 (A), 0 to 1/2 (B), and 0 to 1 (C). Dashed line: TMAO-induced folding of Nank4–7*. The points are experimental data and the lines a global fit to eq 5. The insets show native structures (1rnb (PDB) and full-length/truncated 1ot8 (PDB)) drawn with Chimera. (D) Dependence of the M_w-normalized synergy between urea and TMAO on c_m of the urea-induced unfolding. The letters indicate from which panel the point is taken.

Figure 2

Solvation change of Nank4–7* (A), Nank1–7* (B), and barnase (C) upon unfolding with respect to protein solvation by urea (Δ_UP, orange), TMAO (Δ_TP, red), and water (Δ_WP, blue). The curves were calculated using eqs 8 and 10. Each group of curves represents increasing urea concentration from 0 to 3.5 M in 0.5 M steps (long to short curves). The dashed lines in panel A indicate a solvation pattern that would result in a lack of synergy. Panels D–F show an estimate for the change of TMAO excluded volume upon unfolding vs the radius of gyration of the D state. Distributions/averages for TMAO dihydrate are shown in black/red.

Figure 3

Contributions to the m-values of Nank4–7* (A, B), Nank1–7* (C, D), and barnase (E, F) with respect to urea (right) and TMAO (left). The m-values are given by the bold dashed line and the various additive terms in eq 6 by orange (urea), blue (water), and red (TMAO) lines. Each group of lines of decreasing length represent increasing concentrations of the other respective osmolyte from 0 to 3.3 M (urea) or 2 M (TMAO). (G) Bulk solution terms: Dependence of the chemical activities of urea (U), TMAO (T), and water (W) on the TMAO concentration (γ_ij are defined in eq 7). Note that the solid lines add up to zero, as well as the dashed lines (Gibbs–Duhem relation).