TY  - JOUR
A1  - Borgia, Alessandro
A1  - Zheng, Wenwei
A1  - Buholzer, Karin
A1  - Borgia, Madeleine B.
A1  - Schüler, Anja
A1  - Hofmann, Hagen
A1  - Soranno, Andrea
A1  - Nettels, Daniel
A1  - Gast, Klaus
A1  - Grishaev, Alexander
A1  - Best, Robert B.
A1  - Schuler, Benjamin
T1  - Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods
JF  - Journal of the American Chemical Society
N2  - 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 (R-g). 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 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.
Y1  - 2016
U6  - https://doi.org/10.1021/jacs.6b05917
SN  - 0002-7863
VL  - 138
SP  - 11714
EP  - 11726
PB  - American Chemical Society
CY  - Washington
ER  -