Fig. 5. Model of trimethylamine N-oxide (TMAO), urea and pressure effects on protein folding, based on basic pressure effects (Siebenaller and Somero, 1989), counteracting effects (Yancey et al., 1982) and osmolyte physicochemical studies of Timasheff (1992), Bolen and Baskakov (2000) and Bennion and Daggett (2004). Small spheres represent water molecules. (A) An unfolded protein and/or substrate (S) with hydration layers at a higher density than that of bulk water. (B) Thus, upon folding and/or ligand binding, there is a net expansion (+V) as water molecules are released into bulk water during folding. If this is the case, hydrostatic pressure will inhibit folding and/or binding (A). (C) Addition of urea (U) enhances unfolding since that maximizes favorable binding interactions. In B, TMAO (T) is surrounded by its own structured water layer, which disfavors exposure of the protein's peptide backbone and of the substrate to bulk water. TMAO thus favors folding and binding, reducing the total order (higher in A and C).