Molecular Mechanism for the Denaturation of Proteins by Urea

Almarza, Jorge; Rincón, Luís; Bahsas, Ali; Brito, Francisco

doi:10.1021/bi9007116

Cited by 59 publications

(53 citation statements)

References 47 publications

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“…In the second step, denaturant and water molecules penetrate and solvate the hydrophobic core, resulting in global structural disruption. Experimental support for this model has, however, been scarce and indirect (4,6,13,26).Here, we demonstrate that the well-studied protein Escherichia coli ribonuclease HI (RNase H) (28-30) also rapidly populates a DMG state when exposed to denaturant and use this observation to explore the denaturant dependence of the DMG. We use multisite fluorescence resonance energy transfer (FRET) and kinetic thiol-labeling measurements to dissect the temporal order of protein expansion, side-chain disruption, and water solvation during GdmCl-induced unfolding.…”

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confidence: 85%

“…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).…”

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confidence: 99%

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

Jha

Marqusee

2014

Proc. Natl. Acad. Sci. U.S.A.

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Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).protein unfolding | dry molten globule | steady-state FRET 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 (1-15). In some of these studies, a dry molten globular (DMG) state has been observed on the native side of the free-energy barrier (16-18). The DMG is an expanded form of the native protein in which at least some of the side-chain packing interactions are disrupted without solvation of the hydrophobic core; it was originally postulated to explain heat-induced unfolding of proteins (19,20). Recently, unfolding intermediates resembling the DMG have also been observed in the absence of denaturants, and it has been suggested that the DMG exists in rapid equilibrium with the native state (21-23). The fact that the DMG can be observed by the addition of denaturants is, however, counterintuitive, because traditional models of chemical denaturation rely on changes in solvent-accessible surface area (SASA) (3,24,25) and there is no notable change in SASA during the formation of the DMG.Recent computational studies have suggested an alternative model of chemical denaturation in which denaturants first interact directly with the protein surface, causing the protein to swell, and then penetrate the core (the so-called "direct interaction" mechanism) (9,11,12,26). One of the consequences of this model is that denaturants unfold proteins in two steps (9, 12). In the first step, denaturant molecules displace water molecules within the fir...

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

Jha

Marqusee

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Even in the past year, a number of papers have appeared about whether a chaotrope destabilizes the native state or stabilizes the unfolded state (273)(274)(275)(276). In addition, urea appears to impede the hydrophobic collapse associated with formation of the globular native state (275).…”

Section: Chemical Denaturationmentioning

confidence: 99%

Stability of Protein Pharmaceuticals: An Update

Manning¹,

Chou²,

Murphy³

et al. 2010

Pharm Res

999

782

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In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.

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“…15,19,20 The issue of determination of the specific mode of urea action remains controversial, although the direct mechanism appears to be increasingly favored by the researchers. 17,21,22 The quest for understanding the relative importance of urea interactions with polar versus nonpolar groups is yet another recurrent theme in current investigations.…”

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Urea interactions with protein groups: A volumetric study

2010

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We determined the partial molar volumes and adiabatic compressibilities of N-acetyl amino acid amides, N-acetyl amino acid methylamides, N-acetyl amino acids, and short oligoglycines as a function of urea concentration. We analyze these data within the framework of a statistical thermodynamic formalism to determine the association constants for the reaction in which urea binds to the glycyl unit and each of the naturally occurring amino acid side chains replacing two waters of hydration. Our determined association constants, k, range from 0.04 to 0.39 M. We derive a general equation that links k with changes in free energy, DeltaGtr, accompanying the transfer of functional groups from water to urea. In this equation, DeltaGtr is the sum of a change in the free energy of cavity formation, DeltaDeltaGC, and the differential free energy of solute-solvent interactions, DeltaDeltaGI, in urea and water. The observed range of affinity coefficients, k, corresponds to the values of DeltaDeltaGI ranging from highly favorable to slightly unfavorable. Taken together, our data support a direct interaction model in which urea denatures a protein by concerted action via favorable interactions with a wide range of protein groups. Our derived equation linking k to DeltaGtr suggests that DeltaDeltaGI and, hence, the net transfer free energy, DeltaGtr, are both strongly influenced by the concentration of a solute used in the experiment. We emphasize the need to exercise caution when two solutes differing in solubility are compared to determine the DeltaGtr contribution of a particular functional group.

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Molecular Mechanism for the Denaturation of Proteins by Urea

Cited by 59 publications

References 47 publications

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

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

Stability of Protein Pharmaceuticals: An Update

Urea interactions with protein groups: A volumetric study

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