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

Published: September 14, 2017


Alexander V. Grishaev, Alessandro Borgia, Wenwei Zheng, Karin Buholzer, Madeleine Borgia, Anja Schuler, Hagen Hoffman, Andrea Sorrano, Daniel Nettels, Klaus Gast, Robert Best, Benjamin Schuler


There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded: 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) 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 Forster 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 demonstrate this self-consistency, we have 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 structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.
Citation: Journal of the American Chemical Society
Pub Type: Journals
Created September 14, 2017, Updated February 07, 2018