Aqueous Urea Solutions:  Structure, Energetics, and Urea Aggregation

Stumpe, Martin C.; Grubmueller, Helmut

doi:10.1021/jp066474n

Cited by 210 publications

(224 citation statements)

References 75 publications

Supporting

Mentioning

212

Contrasting

Order By: Relevance

“…Similar H-bonding at both peptide NH and carbonyl sites is reasonable because urea is a very good H-bond donor and acceptor. Urea readily incorporates into the H-bonded network of water and H-bonds equally well with other urea molecules (49)(50)(51)(52)(53). Some MD simulations indicate that urea aggregates in preference to urea-water interaction, but this appears to be an Table 1.…”

Section: Discussionmentioning

confidence: 99%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

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

348

370

View full text Add to dashboard Cite

The mechanism by which urea and guanidinium destabilize protein structure is controversial. We tested the possibility that these denaturants form hydrogen bonds with peptide groups by measuring their ability to block acid-and base-catalyzed peptide hydrogen exchange. The peptide hydrogen bonding found appears sufficient to explain the thermodynamic denaturing effect of urea. Results for guanidinium, however, are contrary to the expectation that it might H-bond. Evidently, urea and guanidinium, although structurally similar, denature proteins by different mechanisms.denaturation ͉ hydrogen exchange ͉ osmolyte ͉ solvation B ecause of its fundamental importance, the study of protein molecular stability has engaged the attention of protein chemists for the last 100 years (1, 2). Experimental and theoretical investigations of protein stability have often focused on small-molecule osmolytes that can be used to modulate stability in vitro (3-6) and are used by virtually all organisms to counter biochemical stress in vivo (7). The mechanism of osmolyte action continues to be controversial. Opposing positions favor either a direct interaction between protein and osmolyte (8-11), such as hydrogen bonding, or an indirect effect mediated by the alteration of water structure (12, 13), or a mixture of both (14)(15)(16)(17)(18)(19). It has been difficult to distinguish between these direct and indirect models because the osmolyte-protein interaction is so weak.It is a thermodynamic truism (20-22) that the concentration of destabilizing osmolytes must be enriched relative to water molecules in the vicinity of the protein surface that becomes newly exposed upon unfolding (preferential interaction). In a thermodynamic sense, denaturing osmolytes ''bind'' selectively to the increased surface of unfolded proteins and thus bias the folded/ unfolded equilibrium toward denaturation. Similarly, stabilizing osmolytes must be preferentially excluded from the protein surface. This is true independently of the physical mechanism of the ''binding'' or ''antibinding'' interaction. Thermodynamically based studies directed at the binding/antibinding problem have been designed to measure the transfer free energy of the various component groups of proteins between water and osmolyte solutions. Results show that the main-chain peptide group makes the largest contribution to the energetics, accounting for Ϸ80% of the effect of urea and of strong stabilizers such as trimethyl amine N-oxide (TMAO) (23). Similar determinations for guanidinium are not yet available, but it seems clear that guanidinium also favorably interacts with the peptide group (8,11,24).To search for the mechanistic basis of thermodynamic destabilization due to urea and guanidinium, we tested the possibility that they exert their denaturing effect through peptide group hydrogen bonding. This can be done experimentally by the straightforward measurement of acid-and base-catalyzed hydrogen exchange (HX) in a small-molecule peptide model. Osmolyte that is H-bonded to the peptide NH ...

show abstract

Section: Discussionmentioning

confidence: 99%

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Lim

Rösgen

Englander

2009

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

348

370

View full text Add to dashboard Cite

show abstract

“…46 In agreement with the preferential interaction with hydrophobic surfaces, urea has also been found to exhibit a moderate tendency to self-associate. 42 Recent molecular dynamics simulations of protein L and lysozyme in mixtures of urea and GdmCl even suggest that this selfaggregation tendency might cause a collapse of unfolded proteins. 36 As the driving force for this collapse, a replacement of urea by guanidinium ions at the polypeptide chain surface has been suggested that results in an enhanced local crowding effect.…”

Section: Discussionmentioning

confidence: 99%

“…2(a)). 41 The precise type of interactions with which denaturants interact with polypeptide chains is still under debate, [42][43][44][45][46] and an empirical binding model is commonly used to describe their effect on the free energy of solvation ( g sol ) of the chain 47 via g sol = −m i ln (1 + K i c i )/n, where m i corresponds to the effective number of binding sites for denaturant molecules, and K i is the apparent equilibrium constant in denaturant i. We note, however, that the assumption of defined binding sites for urea and GdmCl on polypeptide chains is an approximation, since molecular dynamics (MD) simulations have revealed a broad variety of interactions between polypeptide chains, denaturants and water 46 that might affect the dimensions of unfolded proteins.…”

Section: Denaturant Binding In Mixed Denaturantsmentioning

confidence: 99%

Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH

Hofmann

Nettels

Schuler

2013

The Journal of Chemical Physics

View full text Add to dashboard Cite

The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from -3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect. The dimensions of intrinsically disordered and unfolded proteins critically depend on the solution conditions, such as temperature, pH, ionic strength, and osmolyte or denarurant concentration. However, a quantitative understanding of how the complex combination of chain-chain and chain-solvent interactions is affected by the solvent is still missing. Here, we take a step towards this goal by investigating the combined effect of pH and denaturants on the dimensions of an unfolded protein. We use single-molecule fluorescence spectroscopy to extract the dimensions of unfolded cold shock protein (CspTm) in mixtures of the denaturants urea and guanidinium chloride (GdmCl) at neutral and acidic pH. Surprisingly, even though a change in pH from 7 to 2.9 increases the net charge of CspTm from −3.8 to +10.2, the radius of gyration of the chain is very similar under both conditions, indicating that protonation of acidic side chains at low pH results in additional hydrophobic interactions. We use a simple shared binding site model that describes the joint effect of urea and GdmCl, together with polyampholyte theory and an ion cloud model that includes the chemical free energy of counterion interactions and side chain protonation, to quantify this effect. © 2013 AIP Publishing LLC.

show abstract

“…The physical nature of the direct interactions between proteins and urea is also subject of debate, with some authors suggesting that it is mostly electrostatic and related to the formation of direct hydrogen bonds (5,7,12,13), and others suggest that dispersion interactions are the main factor (14,15). Some authors supported the idea the denaturing role of urea is not related to the formation of direct urea-protein interactions, but to its ability to "dry" the protein, weakening the hydrophobic effect responsible for stabilizing protein structures (16)(17)(18)(19). However, recent consensus is that this indirect mechanism is not the main explanation of the effect of urea (18)(19)(20), pointing instead to the direct mechanism.…”

mentioning

confidence: 99%

Toward an atomistic description of the urea-denatured state of proteins

Candotti

Esteban‐Martín

Salvatella

et al. 2013

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

View full text Add to dashboard Cite

We present here the characterization of the structural, dynamics, and energetics of properties of the urea-denatured state of ubiquitin, a small prototypical soluble protein. By combining state-of-the-art molecular dynamics simulations with NMR and small-angle X-ray scattering data, we were able to: (i) define the unfolded state ensemble, (ii) understand the energetics stabilizing unfolded structures in urea, (iii) describe the dedifferential nature of the interactions of the fully unfolded proteins with urea and water, and (iv) characterize the early stages of protein refolding when chemically denatured proteins are transferred to native conditions. The results presented herein are unique in providing a complete picture of the chemically unfolded state of proteins and contribute to deciphering the mechanisms that stabilize the native state of proteins, as well as those that maintain them unfolded in the presence of urea.denaturing mechanism | protein unfolding | random coil | ensemble simulation

show abstract

Aqueous Urea Solutions: Structure, Energetics, and Urea Aggregation

Cited by 210 publications

References 75 publications

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group

Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH

Toward an atomistic description of the urea-denatured state of proteins

Contact Info

Product

Resources

About