Water-Mediated Effects of TMAO on Protein Folding

Trimethylamine N-oxide, commonly denoted as TMAO, is an osmolyte found in marine organisms.  TMAO is known to relieve osmotic stress while combating the denaturing effects of a natural waste product, urea.  Most commonly, the effects of TMAO are attributed to direct binding of the osmolyte to protein structures, thereby removing binding sites for urea.  In contrast, we hypothesize that TMAO reduces the free energy of the bulk solvent, water, and counteracts the unfavorable effects of urea without a necessity for protein binding.  Because a certain number of water molecules are released from the surface of an unfolded protein to the bulk phase upon acquiring a more compact conformation, water should be considered an active participant in the balanced equilibrium reaction. In this project, solubility experiments and calorimetry techniques are used to examine the effects of TMAO on the thermodynamic properties of water.  Diketopiperazine (DKP) is employed as an amide-containing model compound for the backbone of a protein.  DKP solubility was measured as a function of TMAO concentration and pH using a precision density meter to determine the saturation point.  The results indicate a correlation between protein-stabilizing solutions and changes in DKP solubility; increases in TMAO concentration lead to a lowering of DKP solubility.  These findings, along with other results from our laboratory, support the hypothesis that TMAO exerts its favorable effects on protein folding by lowering the free energy of bulk water.