From the Cover: Charge interactions can dominate the dimensions of intrinsically disordered proteins

Abstract

Many eukaryotic proteins are disordered under physiological conditions, and fold into ordered structures only on binding to their cellular targets. Such intrinsically disordered proteins (IDPs) often contain a large fraction of charged amino acids. Here, we use single-molecule Förster resonance energy transfer to investigate the influence of charged residues on the dimensions of unfolded and intrinsically disordered proteins. We find that, in contrast to the compact unfolded conformations that have been observed for many proteins at low denaturant concentration, IDPs can exhibit a prominent expansion at low ionic strength that correlates with their net charge. Charge-balanced polypeptides, however, can exhibit an additional collapse at low ionic strength, as predicted by polyampholyte theory from the attraction between opposite charges in the chain. The pronounced effect of charges on the dimensions of unfolded proteins has important implications for the cellular functions of IDPs.

Fig. 1.

Mean net charge versus mean hydrophobicity per residue of the globular and intrinsically disordered proteins used in this study. The dotted line indicates the separation between intrinsically disordered and globular proteins observed by Uversky et al. (11). Small circles are based on calculations taking into account the amino acid sequence only. Vertical bars indicate the influence of the dye charges; horizontal bars are an estimate of the uncertainty in the hydrophobicity of the dyes. To estimate this uncertainty, the hydrophobicity of the residues at the position of fluorophore attachment was varied between the value for the most hydrophilic and the most hydrophobic amino acid, and the resulting average hydrophobicities were computed. The large circles illustrate this range of values. For CspTm and IN, where the dyes were positioned close to the termini, the entire sequence was used for calculating charge and hydrophobicity, for the ProTα variants only the interdye segment. Hydrophobicity values were calculated according to Kyte and Doolittle (67). The positions used for FRET labeling are indicated as small spheres in the structural representations of the proteins.

Fig. 2.

Single-molecule FRET efficiency (E) histograms of (A) CspTm (C-terminally truncated variant), (B) IN, and (C) ProTαC show the GdmCl dependence of the unfolded proteins. The molar GdmCl concentration is indicated in each panel. (A) The peak at E ≈ 0.95 corresponds to folded CspTm, the peak between E ≈ 0.3 and 0.85 to unfolded protein. (B) First panel: Folded IN (E ≈ 0.9) is only populated in the presence of ZnCl₂ (100 μM; 0 M GdmCl). Other panels: varying concentrations of GdmCl with 1 mM EDTA. 〈E〉 of the unfolded population ranges between 0.4 and 0.6. (C) ProTαC is unfolded under all conditions. For all proteins, the peaks at E ≈ 0 (shaded) correspond to molecules lacking an active acceptor chromophore (68). The solid lines show fits used to extract the mean transfer efficiencies of the subpopulations (17, 23). The dashed lines indicate the mean transfer efficiencies of the unfolded states at the highest GdmCl concentrations.

Fig. 3.

Denaturant-dependent collapse and charge-mediated expansion of unfolded proteins. Dependence of the mean transfer efficiencies, 〈E〉, for CspTm (yellow), IN (red), and ProTαC (blue) on the concentration of GdmCl (filled circles) and urea (open circles). The typical uncertainty in transfer efficiency of individual data points is in the range of 0.02. Error bars are shown for conditions where multiple measurements are available.

Fig. 4.

Dependence of the apparent radii of gyration (R_g) of the labeled protein segments on the concentration of GdmCl (filled circles) and urea (open circles), with (A) CspTm (yellow), (B) IN (red), (C) ProTαN (cyan), and (D) ProTαC (blue). Fits to a binding model for the urea dependence (Eq. 2, colored dashed lines), and to polyampholyte theory for the GdmCl dependence (Eq. 5, black solid lines) are shown. The two components of Eq. 5, corresponding to the contributions of GdmCl binding and electrostatic repulsion, are indicated as continuous and dashed gray lines, respectively. Note that the fits to Eq. 5 are performed based on thermodynamic activities, but plotted on a concentration scale. The colored squares in (A) and (D) indicate the values of R_g on addition of 1 M KCl (compare to Fig. 5). The gray squares indicate the expected values estimated with Eqs. 4 and 5, assuming the values for K, a, and ρ obtained from the fits of the urea dependencies (Table S4), the value of ν obtained from the fits of the GdmCl dependencies (Table S2), and calculating κ for an ionic strength of 1 M. The remaining difference between experimental and calculated values may be due to the preferential interaction of GdmCl with the polypeptide, leading to a higher local charge density than in the bulk solution and a correspondingly stronger charge shielding than for KCl.

Fig. 5.

Effect of charge shielding on denatured state dimensions. Transfer efficiency histograms of CspTm (A) and ProTαC (B) in 1.0 M urea in the absence (Upper) and in the presence of 1.0 M KCl (Lower). CspTm expands on addition of KCl, whereas ProTαC collapses, as predicted by polyampholyte theory. For ProTαC, the change in 〈E〉 corresponds to a reduction in R_g from (4.2 ± 0.2) nm without KCl to (2.98 ± 0.09) nm with KCl, close to the value of (2.87 ± 0.09) nm calculated with Eq. 5 from the corresponding reduction in Debye length. For unfolded CspTm, addition of 1.0 M KCl causes an increase in R_g from (1.73 ± 0.05) nm to (1.97 ± 0.05) nm, with a calculated R_g of the charge-shielded unfolded state of (2.15 ± 0.05) nm.