Exploring the Molecular Mechanism of Trimethylamine‑N‑oxide’s Ability to Counteract the Protein Denaturing Effects of Urea Rahul Sarma and Sandip Paul* Department of Chemistry, Indian Institute of Technology, Guwahati Assam, India-781039 ABSTRACT: Protein denaturation in highly concentrated urea solution is a well-known phenomenon. The counteracting effect of a naturally occurring osmolyte, trimethylamine-N- oxide (TMAO), against urea-conferred protein denaturation is also well-established. However, what is largely unknown is the mechanism by which TMAO counteracts this denaturation. To provide a molecular level understanding of how TMAO protects proteins in highly concentrated urea solution, we report here the structural, energetic, and dynamical properties of N-methylacetamide (NMA) solutions that also contain urea and/or TMAO. The solute NMA is of interest mainly because it contains the peptide linkage in addition to hydrophobic sites and represents the typical solvent-exposed state of proteins. Molecular dynamics computer simulation technique is employed in this study. Analysis of solvation characteristics reveals dehydration of NMA and reduction in hydrogen bond number between NMA oxygen and water upon addition of TMAO. The effect of TMAO on NMA-urea interaction is found to be insignificant. Because TMAO cannot donate its hydrogen to NMA oxygen, the total number of hydrogen bonds formed by NMA oxygen with solution species decreases in the presence of TMAO. In solution, TMAO is found to interact strongly with water and urea. Solvation of TMAO makes the water hydrogen bonding network relatively stronger and reduces relaxation of urea-water hydrogen bonds. Implications of these results for counteracting mechanism of TMAO are discussed. I. INTRODUCTION Natural osmolytes, small organic molecules accumulated by organisms in response to osmotic stress, are known to affect the stability, structure, and function of proteins. In particular, it has been known for many years that a high concentration of urea, which is one of the most commonly available osmolytes, can cause the denaturation of proteins in solution 1 and hence can inhibit many important biological processes. Based on many experimental and theoretical studies, two mechanisms, “indirect” and “direct” interaction models, have been posited for urea-conferred protein denaturation. The indirect mecha- nism presumes that urea acts as a structure breaker for water so as to enhance hydration of protein sites. 2-7 On the other hand, the direct mechanism hypothesizes that urea molecules preferentially bind to protein through their stronger inter- actions with protein backbone or side chains than water. 8-22 Despite the extensive studies from both experiment and theory in the past several decades, it is not clear whether the direct or indirect effect provides the driving force in urea-induced protein denaturation. In all likelihood, urea denaturation of proteins results from combination of both direct and indirect effects. 23-27 In contrast to the protein denaturing effect of urea, some other osmolytes are known to bias the unfolded structure toward the folded state. Trimethylamine-N-oxide (TMAO) represents the extreme among these osmolytes and has generated considerable research interest to the community of biophysicists and biochemists over the past few years. This compound is particularly known for its ability to stabilize proteins 28,29 and nucleic acids, 30 correct medicinally significant issues, such as prion aggregation 31 and cellular folding defects, 32 and counteract protein denaturation by urea, 1,33 heat, and pressure. 34 Although TMAO’s ability to counteract urea- induced protein denaturation at physiological concentrations of urea to TMAO concentration of 2:1 is well-established, 1,33 what is still under debate is the mechanism by which TMAO stabilizes proteins and counteracts urea-conferred protein denaturation. Multiple proposals have been put forward and they range from preferential exclusion of TMAO from the protein 35 through alteration of water structure 36 to preferential solvation of TMAO by water and urea. 37 There is general agreement on the preferential TMAO exclusion from the protein surface. 35,38-42 However, the preferential exclusion model does not explain the role of water in solvation of protein sites. Due to the exclusion of TMAO from the proximity of the protein surface, inclusion of water in the surface of the protein becomes inevitable (termed preferential hydration). If the protein residues are considered to be better hydrated in TMAO solution as compared to pure water or if there is little difference in protein hydration shell between pure water and TMAO solution, then, the question that arises naturally is, why does an unfolded protein shift to its folded state in the presence of TMAO? Clearly, the preferential exclusion model alone is not sufficient to answer this question. Received: February 19, 2013 Revised: April 13, 2013 Article pubs.acs.org/JPCB © XXXX American Chemical Society A dx.doi.org/10.1021/jp401750v | J. Phys. Chem. B XXXX, XXX, XXX-XXX