Structure and Interaction in Aqueous Urea-Trimethylamine-N-oxide Solutions Sandip Paul and Grenfell N. Patey* Contribution from the Department of Chemistry, UniVersity of British Columbia, VancouVer, British Columbia V6T 1Z1, Canada Received November 28, 2006; E-mail: patey@chem.ubc.ca Abstract: The structural and energetic properties of solutions containing water, urea, and trimethylamine- N-oxide (TMAO) are examined using molecular dynamics simulations. Such systems are of interest mainly because TMAO acts to counter the protein denaturing effect of urea. Even at relatively high concentration, TMAO is found to fit well into the urea-water structure. The underlying solution structure is influenced by TMAO, but these perturbations tend to be modest. The TMAO-water and TMAO-urea interaction energies make an important contribution to the total energy in solutions where counter-denaturing effects are expected. TMAO-water and TMAO-urea hydrogen bonds have the largest hydrogen-bond energies in the system. Additionally, TMAO cannot hydrogen bond with itself, and hence it interacts strongly with water and urea. These observations suggest that the mechanism of TMAO counter denaturation is simply that water and urea prefer to solvate TMAO rather than the protein, hence inhibiting its unfolding. I. Introduction Aqueous urea solutions have been of long-standing interest due to their peculiar physical properties. For example, urea increases the solubility of hydrocarbons in water, 1 inhibits micelle formation, 2 and most importantly in high concentration denatures proteins. 3,4 A good deal of effort has been directed toward understanding the mechanism of urea denaturation, but there is still no definitive generally accepted answer to this question, and it remains a subject of active research. Another interesting result concerning chemical denaturation is that the addition of 4 M trimethylamine-N-oxide (TMAO) to 8 M urea solution counteracts the denaturing effect of urea, apparently stablizing the folded state. 5 This observation has movitated recent computer simulation studies, 5 including the present work. Attempts to understand urea denaturation have focused on two concepts that are by no means mutually exclusive. One suggestion is that urea acts indirectly by altering the water structure and consequently the solvation of the denatured protein. A second possibility is that urea stabilizes unfolded states directly by hydrogen bonding with the protein. Recent computer simulations provide at least some evidence for both possibil- ities. 5-8 Bennion and Daggett 6 carried out molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea solution. They report that the water structure is altered by urea, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. Further, because the water structure is weakened by urea, water molecules become free to compete with intraprotein interactions aiding solvation of the unfolded state. Additionally, they note that urea can interact directly with polar groups of the protein, again favoring the denatured state. So, both direct and indirect mechanisms appear to be operative. The existence of both direct and indirect mechanisms of protein denaturation is also supported by the molecular dynamics studies of a ribonuclease A C-peptide analogue reported by Caballero-Herrera et al. 7 We note, however, that both direct and indirect mechanisms are not always equally relevant. For example, Mountain and Thirumalai 8 recently found that direct interaction of urea with site charges was the most important mechanism for the unfolding of hydrocarbon chains in urea-water solution. There have also been simulation studies of aqueous solutions that include urea, TMAO, and proteins. 5,9 Bennion and Daggett 9 investigated the counteraction of urea-induced protein denatur- ation by TMAO. They observed that TMAO enhanced water- water hydrogen bonding both in binary TMAO-water mixtures and in ternary urea-TMAO-water solutions. They report that TMAO has a profound effect on the lifetimes of water-water hydrogen bonds, with 1 M TMAO increasing the lifetime by a factor of ∼3.8 as compared to that of pure water. TMAO also strengthened water-urea interactions and led to a decrease in urea-protein hydrogen bonding. They concluded that the influence of TMAO on the water-water and water-urea interactions is the main factor contributing to its ability to counter urea-induced protein denaturation. In view of their importance, it is of interest to more closely examine the properties of ternary urea-TMAO-water solutions, (1) Wetlaufer, D. B.; Malik, S. K.; Stoller, L.; Coffin, R. L. J. Am. Chem. Soc. 1964, 86, 508. (2) Shick, M. J. J. Phys. Chem. 1964, 68, 3585. (3) Brands, J. F.; Hunt, L. J. J. Am. Chem. Soc. 1967, 89, 4826. (4) Makhatadze, G. I.; Privalov, L. J. J. Mol. Biol. 1967, 226, 491. (5) Daggett, V. Chem. ReV. 2006, 106, 1898 and references therein. (6) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5142. (7) Caballero-Herrera, A.; Nordstrand, K.; Berndt, K. D.; Nilsson, L. Biophys. J. 2005, 89, 842. (8) Mountain, R. D.; Thirumala, D. J. Am. Chem. Soc. 2003, 125, 1950. (9) Bennion, B. J.; Daggett, V. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6433. Published on Web 03/21/2007 4476 9 J. AM. CHEM. SOC. 2007, 129, 4476-4482 10.1021/ja0685506 CCC: $37.00 © 2007 American Chemical Society