Backbone and Side-Chain Contributions in Protein Denaturation by Urea

Canchi, Deepak R.; Garcı́a, Angel E.

doi:10.1016/j.bpj.2011.01.028

Cited by 106 publications

(108 citation statements)

References 56 publications

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“…The side-chain contributions show that urea also interacts favorably with almost all side-chains, the only exceptions being the anionic aspartate and glutamate residues, consistent with an earlier study 8 . Our results thus suggest that both backbone and side-chains contribute comparable amounts to the favorable solvation of the unfolded state by urea solutions, in agreement with the results of other recent computational studies 7, 92 . Note that this does not mean they contribute equally to folding m -values, which measure how the difference between the folded and unfolded Δμ tr changes with denaturant concentration.…”

Section: Resultssupporting

confidence: 92%

“…That is, Γ UP measures how much urea must be added to keep the bulk urea chemical potential μ U constant when a protein is added to the solution and is expected to be positive if urea interacts favourably with the protein, and vice versa. In simulations, the coefficient can be estimated very simply from the heuristic relation: 89–92

Γ_{UP} = false〈 n_{U}^{P} - n_{W}^{P} true(\frac{n_{normalU}^{normalB}}{n_{normalW}^{normalB}} true) false〉

In this equation,

n_{U}^{P}

and

n_{W}^{P}

are the number of urea and water molecules in a defined volume close to the protein, while

n_{U}^{B}

and

n_{W}^{B}

are the corresponding numbers in the bulk solution away from the protein, i.e. Γ UP is the average number of urea molecules in the volume near the protein, in excess of what would be expected based on the bulk solution composition.…”

Section: Resultsmentioning

confidence: 99%

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Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment

et al. 2016

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Chemical denaturants are the most commonly used agent for unfolding proteins, and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation, with studies using Förster resonance energy transfer (FRET) and other methods finding an increase in radius of gyration with denaturant concentration, but with small-angle X-ray scattering (SAXS) studies finding mostly no change. This discrepancy therefore challenges our understanding of denaturation mechanism, and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favourable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET transfer efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely that the commonly used FRET chromophores do not qualitatively alter the results, and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.

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Section: Resultssupporting

confidence: 92%

Γ_{UP} = false〈 n_{U}^{P} - n_{W}^{P} true(\frac{n_{normalU}^{normalB}}{n_{normalW}^{normalB}} true) false〉

In this equation,

n_{U}^{P}

and

n_{W}^{P}

are the number of urea and water molecules in a defined volume close to the protein, while

n_{U}^{B}

and

n_{W}^{B}

Section: Resultsmentioning

confidence: 99%

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment

et al. 2016

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“…Urea is a denaturing osmolyte; it favors unfolded and extended protein conformations through a combination of preferential interactions with the protein backbone and side chains [76-81]. Urea may also exert an indirect effect on protein stability via alterations in the water network surrounding the protein [82].…”

Section: Resultsmentioning

confidence: 99%

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction

Yarawsky

English

Whitten

et al. 2017

Journal of Molecular Biology

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Staphylococcus epidermidis is one of the primary bacterial species responsible for healthcare-associated infections. The most significant virulence factor for S. epidermidis is its ability to form a biofilm, which renders the bacteria highly resistant to host immune responses and antibiotic action. Intercellular adhesion within the biofilm is mediated by the accumulation-associated protein (Aap), a cell wall-anchored protein that self-assembles in a zinc-dependent manner. The C-terminal portion of Aap contains a proline/glycine-rich, 135 amino acid-long region that has not yet been characterized. The region contains a set of 18 nearly identical AEPGKP repeats. Analysis of the proline/glycine-rich region (PGR) using biophysical techniques demonstrated the region is a highly extended, intrinsically disordered polypeptide (IDP) with unusually high polyproline type II helix (PPII) propensity. In contrast to many IDPs, there was a minimal temperature dependence of the global conformational state of PGR in solution as measured by analytical ultracentrifugation and dynamic light scattering. Furthermore, PGR was resistant to conformational collapse or α-helix formation upon addition of the osmolyte TMAO or the cosolvent TFE. Collectively, these results suggest PGR functions as a resilient, extended stalk that projects the rest of Aap outward from the bacterial cell wall, promoting intercellular adhesion between cells in the biofilm. This work sheds light on regions of low complexity often found near the attachment point of bacterial cell wall-anchored proteins.

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“…A computational treatment of aqueous urea solutions, developed by refining molecular dynamics simulation parameters until urea-urea and urea-water Kirkwood-Buff (KB) integrals were reproduced (32), was incorporated in simulations to predict changes in urea-protein preferential interaction coefficients and urea m-values for folding/unfolding the trp-cage miniprotein in urea solutions (33). Preferential interactions of urea with sidechain and backbone groups exposed in unfolding were found to contribute 60% and 40% of the urea m-value, respectively.…”

Section: Why Urea Destabilizes and Gb Stabilizes Globular Proteins: Amentioning

confidence: 99%

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

Guinn

Pegram²,

Michael³

et al. 2011

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

220

431

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To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K p for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K p values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH-amide O and amide NH-amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K p values. U rea and glycine betaine (GB) rank at opposite ends of a series of small nonelectrolyte solutes in terms of their effects on protein folding and other protein processes. Stabilities (ΔG o obs ) of folded proteins and of site-specific protein-DNA complexes decrease linearly with increasing urea molarity and increase with increasing GB molarity (1-5). This solute series parallels the Hofmeister anion and cation series of non-Coulombic effects of salt ions on protein processes; guanidinium cation and thiosulfate or iodide anions are highly destabilizing but alkali metal cations are not destabilizing and sulfate or fluoride anions are stabilizing (6-8). Similar rank orders but smaller ranges of solute and nonCoulombic salt effects are observed on DNA and RNA duplex formation; urea and salt ions that greatly destabilize proteins when added at molar concentrations also greatly destabilize DNA duplexes, but GB and Hofmeister salt ions that stabilize proteins do not stabilize nucleic acids duplexes (4,5,(8)(9)(10)(11). Another range of solute effects is observed for the series of solutes from ethylene glycol (EG) to PEG, where the monomer EG destabilizes both hairpin and duplex DNA helices, whereas polymeric PEGs greatly stabilize the duplex and eliminate the destabilization of the hairpin helix (12). In our research, we use molecular thermodynamic analyses of model compound data to interpret and predict the effects of these solutes an...

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Backbone and Side-Chain Contributions in Protein Denaturation by Urea

Cited by 106 publications

References 56 publications

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

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