title: Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.
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.
authors: Alessandro
authors: A
authors: Borgia
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
authors: Wenwei
authors: W
authors: Zheng
authors: Karin
authors: K
authors: Buholzer
authors: Madeleine B
authors: MB
authors: Borgia
authors: Physical Biochemistry, University of Potsdam , 14476 Potsdam, Germany.
authors: Anja
authors: A
authors: Schüler
authors: †Department of Biochemistry and #Department of Physics, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors: Hagen
authors: H
authors: Hofmann
authors: Andrea
authors: A
authors: Soranno
authors: †Department of Biochemistry and #Department of Physics, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors: Daniel
authors: D
authors: Nettels
authors: Physical Biochemistry, University of Potsdam , 14476 Potsdam, Germany.
authors: Klaus
authors: K
authors: Gast
authors: National Institute of Standards and Technology and the Institute for Bioscience and Biotechnology Research, University of Maryland , Rockville, Maryland 20850, United States.
authors: Alexander
authors: A
authors: Grishaev
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892-0520, United States.
authors: Robert B
authors: RB
authors: Best
authors: Benjamin
authors: B
authors: Schuler
mesh_terms: D001499:Bayes Theorem; D031541:Fluorescence Resonance Energy Transfer; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D010455:Peptides