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J Am Chem Soc. 2016 Sep 14;138(36):11714-26. doi: 10.1021/jacs.6b05917. Epub 2016 Sep 1.

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

Author information

1
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
2
Physical Biochemistry, University of Potsdam , 14476 Potsdam, Germany.
3
†Department of Biochemistry and #Department of Physics, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
4
National Institute of Standards and Technology and the Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, Maryland 20850, United States.

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.

PMID:
27583570
PMCID:
PMC5597961
DOI:
10.1021/jacs.6b05917
[Indexed for MEDLINE]
Free PMC Article

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