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

Wenwei Zheng, Alessandro Borgia, Karin Buholzer, Alexander Grishaev, Benjamin Schuler, Robert B. Best

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Chemical denaturants are the most commonly used agents 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. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found 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 favorable 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 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.

Original languageEnglish (US)
Pages (from-to)11702-11713
Number of pages12
JournalJournal of the American Chemical Society
Volume138
Issue number36
DOIs
StatePublished - Sep 14 2016
Externally publishedYes

Fingerprint

Intrinsically Disordered Proteins
Pharmacologic Actions
Energy Transfer
Proteins
Denaturation
Guanidine
X-Rays
Experiments
X ray scattering
Energy transfer
Urea
Association reactions
Protein Unfolding
Hydrogen Bonding
Solvation
Chromophores
Water
Free energy
Hydrogen bonds
Trajectories

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment. / Zheng, Wenwei; Borgia, Alessandro; Buholzer, Karin; Grishaev, Alexander; Schuler, Benjamin; Best, Robert B.

In: Journal of the American Chemical Society, Vol. 138, No. 36, 14.09.2016, p. 11702-11713.

Research output: Contribution to journalArticle

Zheng, Wenwei ; Borgia, Alessandro ; Buholzer, Karin ; Grishaev, Alexander ; Schuler, Benjamin ; Best, Robert B. / Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment. In: Journal of the American Chemical Society. 2016 ; Vol. 138, No. 36. pp. 11702-11713.
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