The effect of denaturants on protein structure

Abstract

Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. J Mol B i d 226491-505).Keywords: denaturants; dihydrofolate reductase; protein mobility; ribonuclease A Despite our routine use of them, our understanding of the molecular mechanism by which chemical denaturants like urea or guanidinium (Gua) cause a protein to unfold is still rather limited. As has been pointed out by others (Schellman, 1987), part of the problem is that it is difficult to measure binding constants when the protein concentration is M and the denaturant concentration is 4 M. High precision scanning calorimetry has provided one experimental solution to this problem (Makhatadze & Privalov, 1992) but relating the thermodynamic data produced in such an experiment to protein structure can be difficult (Alber, 1989). While it does seem clear that denaturants interact directly with the protein (Simpson & Kauzmann, 1953), it is also clear that at high concentrations these molecules can cause substantial changes to the behavior of the solvent itself (Breslow & Guo, 1990 teins by migrating into the interior of the protein and forming hydrogen bonds to atoms in the backbone (Hedwig et al., 1991). In support of such a model, the exchange rates of some peptide NH protons, which are found on both the surface and the interior of a protein, are decreased in the presence of urea (Kim & Woodward, 1993). In addition, crystallographic studies of diketopiperazine and urea show hydrogen bonding between the amide groups and the urea (Thayer et al., 1993). Other models have suggested that denaturants act by decreasing the hydrophobic effect (Wetlaufer et al., 1964;Roseman & Jencks, 1975), which, in this instance, is usually postulated to be the dominant force stabilizing protein structure. Finally, experiments by Kim and Woodward (1 993) clearly show that, in addition to interacting with peptide bonds, urea alters...

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

“…5B). Low concentrations of guanidine hydrochloride enhance the fluorescence of the domain (Ϯ metal ions), suggesting a possible interaction between GdnHCl and the DNase that has been documented for other proteins (35). The fluorescence of the fully denatured protein was the same regardless of its metal loading, indicating that the fluorescence of the unfolded states of the apo-and holoenzyme are the same.…”

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride

“…The change in heat capacity of unfolding measured with heat unfolding and denaturant-induced unfolding are different, indicating that chemical denaturant may participate directly in the process (41). Discrete denaturant binding sites and low B-factors of surface residues have also been observed in the X-ray crystal structures of proteins in the presence of GdmCl (7,8,42).…”

“…D enaturants such as guanidinium chloride (GdmCl) and urea are classic perturbants used to probe the thermodynamics and kinetics of protein conformational changes, although their mechanism of action is poorly understood (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In some of these studies, a dry molten globular (DMG) state has been observed on the native side of the free-energy barrier (16)(17)(18).…”

Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

“…55,56 In the case of cyt c, Thomas et al 48 proposed that a guanidinium ion may displace the side-chain of Arg38 in cyt c, which forms a functionally important salt bridge involving the buried heme propionate side-chain. 57 To address this possibility, we carried out a detailed comparison of the GuHCl and urea-induced unfolding transitions for H33N cyt c. In the absence of specific binding effects, we expect that the free energy of unfolding extrapolated to cZ0 is independent of the denaturant used.…”