Conformational Ensemble of Disordered Proteins Probed by Solvent Paramagnetic Relaxation Enhancement (sPRE)

Abstract

Characterization of the conformational ensemble of disordered proteins is highly important for understanding protein folding and aggregation mechanisms, but remains a computational and experimental challenge owing to the dynamic nature of these proteins. New observables that can provide unique insights into transient residual structures in disordered proteins are needed. Here using denatured ubiquitin as a model system, NMR solvent paramagnetic relaxation enhancement (sPRE) measurements provide an accurate and highly sensitive probe for detecting low populations of residual structure in a disordered protein. Furthermore, a new ensemble calculation approach based on sPRE restraints in conjunction with residual dipolar couplings (RDCs) and small-angle X-ray scattering (SAXS) is used to define the conformational ensemble of disordered proteins at atomic resolution. The approach presented should be applicable to a wide range of dynamic macromolecules.

Keywords: NMR spectroscopy; ensemble calculations; intrinsically disordered proteins; paramagnetic relaxation enhancement; protein dynamics.

Figure 1.

sPRE studies on denatured ubiquitin. a) ¹H, ¹⁵N HSQC spectra of ubiquitin denatured in 8 M urea at pH 2.5 in the absence (left, black) and presence (right, red) of 4 mM Gd-DTPA-BMA. b) Overlay of 1D projections of the spectra shown in panel a. c) Correlation of sPRE data measured in the presence of 1 mM and 2 mM Gd-DTPA-BMA. The correlation coefficient (r) is shown. d) Transverse proton relaxation rates (¹H^N-R₂) plotted against Gd-DTPA-BMA concentrations for selected residues in urea-denatured ubiquitin. The plot shows that the change in relaxation rate as a function of paramagnetic probe concentration is linear. The slope of this line corresponds to the relaxivity for each residue (in units of mM⁻¹s⁻¹), which is defined as the change in relaxation rate normalized to the probe concentration.

Figure 2.

sPRE reveals native-like structure in denatured ubiquitin. a) Transverse amide proton sPRE rates (¹H^N-Γ₂) of urea-denatured ubiquitin obtained in the presence of 2 mM Gd-DTPA-BMA plotted against the residue number. Secondary structure elements of native ubiquitin are shown above the plot and the corresponding regions are shaded. b) The zoomed-in view shows sPRE data points for residues T12-E16 (with the sequence TITLE) that correspond to a β-hairpin in native ubiquitin (PDB: 1D3Z, shown as cartoon). Amide protons of residues in the β2 strand that are involved in hydrogen-bonding with the opposite strand display lower sPRE values than neighboring residues. Backbone atoms in the β2 strand are shown as sticks with C, H, N and O colored as cyan, gray, blue and red, respectively.

Figure 3.

Convergence of the fit to experimental data by the increase in ensemble size. Data were obtained by simulated annealing calculations of denatured ubiquitin at different ensemble sizes using three sets of RDCs (H-N, Cα-Hα, Cα-C’), sPRE and SAXS data as structural restraints. An ensemble size of ~5 (equally weighted members) is sufficient to satisfy all experimental data within experimental error.

Figure 4.

Cα-Cα contact maps of urea-denatured ubiquitin from an ensemble of 10,000 conformers (2000 structures with an ensemble size of 5) obtained from Xplor-NIH simulated annealing calculations without experimental restraints (left), with RDC/SAXS (middle) and sPRE/RDC/SAXS (right) restraints. Contacts are color-coded according to contact population (probability), defined as the total number of Cα-Cα contacts within 8 Å distance cut-off divided by the total number of conformers. Native contacts, i.e. observed in the NMR structure of ubiquitin (PDB: 1D3Z), are enclosed by green lines. For clarity, only contacts between residues more than 3 residues apart (|i-j|>3) are shown.