Distribution of Charged Residues Affects the Average Size and Shape of Intrinsically Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

Keywords: average shape of conformational ensembles; charge clustering; charged-residue patterning; conformational compactness; ellipsoid model; hydrodynamic radius; polyelectrolytes; proline content; solvent-accessible surface area.

Figure 1

Scheme of the experimental plan used in this work. (a) Scheme of the primary structures of a protein set, derived from a generic wild-type IDP by distributing more evenly the oppositely charged residues (low-κ variant) or by clustering them in two blocks at the N- and C-moieties (high-κ variant). Only charged residues were permutated, preserving the original location in the sequence of non-charged residues (see also Figure S1). Blue and red spheres indicate positively and negatively charged residues, respectively. Gray spheres indicate all the other amino acid residues. (b) The conformational ensemble of each model IDP was investigated by size-exclusion chromatography (SEC) and native mass spectrometry (MS), to derive experimental values of R_h and SASA. (c) R_h and SASA values were combined to calculate the volume and depict the average shape from the ensemble of each model IDP.

Figure 2

Comparative bioinformatic analyses of N_TAIL (a), NFM (b) and PNT4 (c). Upper panels: The FCR, fraction of charged residues, was calculated by CIDER [32]. Each model protein contains charged residues at high density, with red and blue bars indicating negative and positive charges, respectively. The increase in κ value is reflected in the progressively more “blocky” distribution of charged residues. Lower panel: each protein is predicted to be predominantly disordered by IUPred [33]. The discrepancy from the disorder threshold value (0.5) in the IUPred score is shaded in gray. The IUPred and CIDER outputs were generated using the default options of the respective web server.

Figure 3

Compactness of the model IDPs. (a) CI derived from the R_h (CI_R); (b) CI derived from the average SASA of the conformational ensemble (${\overline{CI}}_{SASA}$) of N_TAIL, NFM and PNT4 variants (Lκ: low-κ; wt: wild type; Hκ: high-κ). Mean values of three independent measurements are shown with error bars indicating standard deviations. Statistical analyses were carried out using Welch’s t-test, n.s.: not significant p > 0.05, *: p < 0.05, **: p < 0.01.

Figure 4

Native-MS analyses. (a) NanoESI-MS spectra of N_TAIL variants acquired under non-denaturing conditions (50 mM ammonium acetate pH 7.0). The most intense signal of each peak-envelope is labeled by the corresponding charge state. (b–d) Multi-Gaussian deconvolution of the MS spectra obtained for N_TAIL (b), NFM (c) and PNT4 (d), in the low-κ (upper row), wt (central row) and high-κ (bottom row) variants. Extended (Ext), intermediate (Int) and compact (Com) species are colored with different shades and labeled in the upper panels. MS spectra of NFM and PNT4 variants are reported in Figure S3.

Figure 5

Relationship between ellipsoid volume and κ values. (a) Regression of ellipsoid volume and κ for N_TAIL (light blue), NFM (orange) and PNT4 (green). The equation of trend lines are: y = −33.4 x + 51.7, R² = 0.987 for N_TAIL, y = −61.2 x + 101.4, R² = 0.998 for NFM and y = 4.3 x + 27.2, R² = 0.710 for PNT4. Mean values of three independent measurements are represented, with error bars indicating standard deviations. (b) Geometry of the model proteins as obtained by applying the ellipsoid model.