Molecular dynamics simulations of the protein chymotrypsin inhibitor 2 in 8 M urea at 60°C were undertaken to investigate the molecular basis of chemical denaturation. The protein unfolded rapidly under these conditions, but it retained its native structure in a control simulation in water at the same temperature. The overall process of unfolding in urea was similar to that observed in thermal denaturation simulations above the protein's Tm of 75°C. The first step in unfolding was expansion of the hydrophobic core. Then, the core was solvated by water and later by urea. The denatured structures in both urea and at high temperature contained residual native helical structure, whereas the ␤-structure was completely disrupted. The average residence time for urea around hydrophilic groups was six times greater than around hydrophobic residues and in all cases greater than the corresponding water residence times. Water self-diffusion was reduced 40% in 8 M urea. Urea altered water structure and dynamics, thereby diminishing the hydrophobic effect and encouraging solvation of hydrophobic groups. In addition, through urea's weakening of water structure, water became free to compete with intraprotein interactions. Urea also interacted directly with polar residues and the peptide backbone, thereby stabilizing nonnative conformations. These simulations suggest that urea denatures proteins via both direct and indirect mechanisms.S mall organic molecules in aqueous solution can have profound effects on protein stability, structure, and function. The use of these solutions to stabilize or destabilize proteins, depending on the cosolvent, is commonplace. In fact, protein studies are conducted almost exclusively in complex solutions. Chemical denaturation, with an agent such as urea, is one of the primary ways to assess protein stability, the effects of mutations on stability, and protein unfolding (1). Despite its widespread use, the molecular basis for urea's ability to denature proteins remains unknown. Urea may exert its effect directly, by binding to the protein, or indirectly, by altering the solvent environment (2-20). Most versions of the direct interaction model posit that urea binds to, and stabilizes, the denatured state (D), thereby favoring unfolding. But this interpretation does not explain how the protein surmounts the kinetic barrier to unfolding. In this regard, urea could bind to the protein and compete with native interactions, thereby actively participating in the unfolding process. Alternatively, it has been proposed that urea acts indirectly by altering the solvent environment, thereby mitigating the hydrophobic effect and facilitating the exposure of residues in the hydrophobic core. It is also possible that the mechanism of urea-promoted unfolding depends on the urea concentration. Unfortunately, it seems unlikely that experimental approaches will provide the molecular details of how urea denatures proteins, so we are employing atomic-resolution molecular dynamics (MD) simulations to address this issue....

Trimethylamine n-oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins and offsets the destabilizing effects of urea. To investigate the molecular mechanism of these effects, we have studied the thermodynamics of interaction between TMAO and protein functional groups. The solubilities of a homologous series of cyclic dipeptides were measured by differential refractive index and the dissolution heats were determined calorimetrically as a function of TMAO concentration at 25 degrees C. The transfer free energy of the amide unit (-CONH-) from water to 1 M TMAO is large and positive, indicating an unfavorable interaction between the TMAO solution and the amide unit. This unfavorable interaction is enthalpic in origin. The interaction between TMAO and apolar groups is slightly favorable. The transfer free energy of apolar groups from water to TMAO consists of favorable enthalpic and unfavorable entropic contributions. This is in contrast to the contributions for the interaction between urea and apolar groups. Molecular dynamics simulations were performed to provide a structural framework for the interpretation of these results. The simulations show enhancement of water structure by TMAO in the form of a slight increase in the number of hydrogen bonds per water molecule, stronger water hydrogen bonds, and long-range spatial ordering of the solvent. These findings suggest that TMAO stabilizes proteins via enhancement of water structure, such that interactions with the amide unit are discouraged.

Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO͞8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.M echanisms have evolved in nature to allow living organisms to compensate for extreme conditions. For example, certain marine creatures have adapted to life at high pressures and salinity by using osmolytes to maintain cellular volume and buoyancy (1, 2). However, certain osmolytes, like urea, can degrade protein function and disrupt their structures at the high concentrations found in some animals and marine life (1-3), although elevated pressures (Ϸ70 MPa) could mitigate some of the deleterious effects of urea (4-6). Nevertheless, the answer to this paradox was the discovery of protective osmolytes such as betaine and trimethylamine N-oxide (TMAO) in certain elasmobranchs by Yancey et al. (1,[7][8] and later in other marine organisms and mammals (1,6,9). In marine animals, the TMAO concentration varies with habitat depth, presumably as a response to pressure (4-5). Furthermore, in organisms that concentrate urea as an osmolyte (7) and buoyancy factor (2), TMAO has been found in urea at ratios of 3:1 and 2:1 (7, 10).TMAO can restore enzyme function that has been lost because of the presence of urea (6, 10-12) by restoring the protein to its native structure (13-15). The mechanism of action of these protective osmolytes is not understood fully; both direct (16)(17)(18)(19) and indirect (13,(19)(20)(21) interactions have been proposed, and the mechanism may be molecule-specific (14). Our understanding of the mechanism of action of chemical denaturants, such as urea and guanidinium chloride, is in a similar state (19,20,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Consequently, we are pursuing molecular dynamics (MD) simulations of such compounds in an attempt to characterize these mechanisms at the molecular level.Here, we investigate the ability of TMAO to overcome the effect of urea on protein structure at the atomic level. Chymotrypsin inhibitor 2 (CI2) was chosen for this study because of the extensive amoun...

We have traditionally relied on extremely elevated temperatures (498 K, 225 8C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298 K to 498 K. The simulation lengths vary from 94 ns to 20 ns, for a total simulation time of 344 ns, or 0.34 ms. At 298 K, the protein is very stable over the full 50 ns simulation. At 348 K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25 ns, explores partially unfolded conformations for 20 ns, and then refolds over the last 35 ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498 K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.