The molecular basis for the chemical denaturation of proteins by urea

Abstract

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....

Counteraction of urea-induced protein denaturation by trimethylamine N -oxide: A chemical chaperone at atomic resolution

“…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.…”

“…45). The simulations of CI2 in water at 125°C and in 8 M urea have been described (22,40). The control trajectory of CI2 in pure water at 60°C was prepared for MD as described (22), yielding 2,596 water molecules.…”

“…The simulations of CI2 in water at 125°C and in 8 M urea have been described (22,40). The control trajectory of CI2 in pure water at 60°C was prepared for MD as described (22), yielding 2,596 water molecules.We constructed 8 M-urea systems (mole fraction of 0.186) as described (22). Briefly, water molecules were replaced randomly with urea, resulting in 2,103 water molecules and 493 urea molecules.…”

Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

“…39 It is also possible that decreasing pH interferes with the solubility of the protein in urea. 40 This technique provides a method for more detailed study into the mechanisms of BM assembly.…”

Characterizing folding, structure, molecular interactions and ligand gated activation of single sodium/proton antiporters

“…Denaturants of chemical (urea, Gdn-HCl, pH) (Anderson et al 1990;Alonso and Dill 1991;Bennion and Dagget 2003) or physical (high temperature, pressure) (Makhatadze and Privalov 1992;Silva and Weber 1993;Sturtevant 1987) natures change interactions within a protein finally leading to the loss of its functional structure. Alterations of molecular conformations change macroscopic properties of the molecular assembly which can be observed for example as changes in their fluorescence, heat capacity, and their viscosity.…”