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) 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 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 comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations

“…With increasing GdmCl concentration, the unfolded state is increasingly populated, and its peak shifts from higher to lower transfer efficiency (Fig. 2B), reflecting chain expansion, as observed previously for protein L (37-39) and many other proteins (23,40,41).…”

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

“…This artifact is widely acknowledged, and it has been proposed that using a self-avoiding walk (SAW) model is a better description for protein chains at high denaturant concentrations, where they are typically close to the EV limit [28][29][30][31] . In a recent study of an unfolded and an intrinsically disordered protein in chemical denaturants, we indeed found that applying a SAW model to FRET data at high denaturant concentrations led to inferred average properties ( and ) close to those obtained by a global analysis integrating both FRET and SAXS data in an ensemble description based on atomistic simulations, while the result from using a Gaussian chain overestimated these properties 4 . On the other hand, at low denaturant concentrations, and inferred from a Gaussian chain model were closer to those from a global analysis than the SAW model.…”

“…Studying the structure, dynamics and function of these disordered polypeptide chains, as well as other disordered biopolymers, requires new experimental methods, and new methods of interpreting the data, since they are inherently averaged over a broad ensemble of heteropolymer configurations [2][3][4] . Examples of experiments which can help address this challenge include: nuclear magnetic resonance (NMR), which can provide short-range structural information via scalar coupling, chemical shift and NOE data 5 , as well as long-range information by paramagnetic relaxation enhancement (PRE) measurements 6 , and global information from diffusion coefficient measurements; light-scattering 7 and two-focus FCS 4,8 , which also yield diffusion coefficients and hence hydrodynamic radius; small-angle X-ray (or neutron) scattering (SAXS or SANS), which directly gives information on inter-and intramolecular pair distance distributions 9 ; and Förster resonance energy transfer (FRET), in particular single-molecule FRET, which probes the distribution of distances between pairs of chromophore labels attached to the molecule of interest, as well as the associated dynamics, and often enables structured and unstructured subpopulations to be separated 10,11 . Obtaining as detailed as possible a picture of the disordered ensemble would ideally combine information from all of these experiments, if available.…”

“…Obtaining as detailed as possible a picture of the disordered ensemble would ideally combine information from all of these experiments, if available. This can be done most comprehensively via an explicit ensemble description of the disordered state, either by reweighting of an existing molecular simulation [2][3][4][12][13][14][15][16][17] , or by performing an ensemble structural refinement with a very large number of replicas of the system [18][19][20][21][22][23] . However, a simpler approach is frequently useful.…”

Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS

“…Our results favor the former, but the extent of collapse can of course depend on the solution conditions and the net charge of the chain (53,82). Resolving this controversy will require further concerted efforts involving a direct comparison of the different methods, either using time-resolved ensemble methods or proteins that remain unfolded even at low denaturant concentrations (83,84).…”