Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods

Alessandro Borgia, Wenwei Zheng, Karin Buholzer, Madeleine B. Borgia, Anja Schüler, Hagen Hofmann, Andrea Soranno, Daniel Nettels, Klaus Gast, Alexander Grishaev, Robert B. Best, Benjamin Schuler

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Original languageEnglish (US)
Pages (from-to)11714-11726
Number of pages13
JournalJournal of the American Chemical Society
Volume138
Issue number36
DOIs
StatePublished - Sep 14 2016
Externally publishedYes

Fingerprint

Polypeptides
X ray scattering
Energy Transfer
X-Rays
Intrinsically Disordered Proteins
Energy transfer
Proteins
Peptides
Dynamic light scattering
Protein folding
Spectrin
Protein Stability
Fluorescence Spectrometry
Protein Folding
Staphylococcal Protein A
Hydrodynamics
Fluorescence
Spectroscopy
Molecules
Dynamic Light Scattering

ASJC Scopus subject areas

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

Cite this

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods. / Borgia, Alessandro; Zheng, Wenwei; Buholzer, Karin; Borgia, Madeleine B.; Schüler, Anja; Hofmann, Hagen; Soranno, Andrea; Nettels, Daniel; Gast, Klaus; Grishaev, Alexander; Best, Robert B.; Schuler, Benjamin.

In: Journal of the American Chemical Society, Vol. 138, No. 36, 14.09.2016, p. 11714-11726.

Research output: Contribution to journalArticle

Borgia, A, Zheng, W, Buholzer, K, Borgia, MB, Schüler, A, Hofmann, H, Soranno, A, Nettels, D, Gast, K, Grishaev, A, Best, RB & Schuler, B 2016, 'Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods', Journal of the American Chemical Society, vol. 138, no. 36, pp. 11714-11726. https://doi.org/10.1021/jacs.6b05917
Borgia, Alessandro ; Zheng, Wenwei ; Buholzer, Karin ; Borgia, Madeleine B. ; Schüler, Anja ; Hofmann, Hagen ; Soranno, Andrea ; Nettels, Daniel ; Gast, Klaus ; Grishaev, Alexander ; Best, Robert B. ; Schuler, Benjamin. / Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods. In: Journal of the American Chemical Society. 2016 ; Vol. 138, No. 36. pp. 11714-11726.
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