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

Abstract

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), smallangle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRETbased nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.protein folding | unfolded protein ensemble | Förster resonance energy transfer | nuclear magnetic resonance | small-angle X-ray scattering P roteins exist as ensembles of interconverting conformations.Obviously, unfolded polypeptide chains, such as chemically or physically denatured proteins and intrinsically disordered proteins (IDPs), can access extremely large numbers of conformations (1). A comprehensive description of their structural and dynamical behavior is a prerequisite for understanding protein folding (2-4) and the function of IDPs in health and disease (5, 6). Due to the very large number of conformational degrees of freedom of the unfolded polypeptide ensemble, it is of utmost importance to obtain as many independent experimental parameters as possible for a quantitative description of its behavior. Three experimental methods have been particularly informative in this respect: NMR spectroscopy (2, 5, 7-9), small angle X-ray scattering (SAXS) (10, 11), and single-molecule Förster resonance energy transfer spectroscopy (single-molecule FRET) (12, 13).NMR in solution provides very rich local structural and dynamical information at virtually any atom site with the exception of oxygen. Distance and angular information can be obtained from nuclear Overhauser enhancements (14), three-bond scalar couplings (15), paramagnetic relaxation enhancements (PREs) (16), pseudo contact shifts (17), residual dipolar couplings (RDCs) (8, 18), chemical shifts (19), and hydrogen bond scalar ...

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements

“…Their results point to consistent inferences for average distances and distributions of distances for ubiquitin in high concentrations of denaturant (65). A similar consistency regarding denaturant-mediated expansion was reported by Borgia et al (23), who used a combination of smFRET, SAXS, dynamic light scattering, and two-focus fluorescence correlation spectroscopy to assess how conformational ensembles change as a function of denaturant concentration.…”

“…Aznauryan et al (65) performed SAXS and smFRET measurements and combined these with distances extracted from structural ensembles based on data from NMR experiments. Their results point to consistent inferences for average distances and distributions of distances for ubiquitin in high concentrations of denaturant (65).…”

Inferring Properties of Disordered Chains From FRET Transfer Efficiencies

“…In fact, application of the SAW- model to a poly-Val homopolymer yields results of similar accuracy for R and R g as those obtained for the natural protein sequences. Of course, a pronounced patterning of sequence properties as found in some IDPs the positions of FRET donor and acceptor in the protein and seeing whether they can be described globally with a single set of fit parameters 4,28,73,74 . Discrepancies would suggest that a homopolymer P(r) cannot provide a self-consistent interpretation of the data and that more detailed analysis is needed, including, e.g., atomistic simulations or ensemble reweighting, ideally combined with additional segment-specific experimental information, e.g.…”

“…Discrepancies would suggest that a homopolymer P(r) cannot provide a self-consistent interpretation of the data and that more detailed analysis is needed, including, e.g., atomistic simulations or ensemble reweighting, ideally combined with additional segment-specific experimental information, e.g. from NMR 3,74 .…”

Simulation of FRET dyes allows quantitative comparison against experimental data

“…Now we determine an effective segment length N eff = N + L with a "length" L of the dye pair which was already used to interpret experimental FRET measurements. 66,67 We take the dyes' mean square separation R 2 DA 1/2 and assign an N eff from the respective fit. An example fit and details of the calculation are given in the supplementary material (see Fig.…”