The Complex Energy Landscape of the Protein IscU

Abstract

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural information from NMR and SAXS suggests that the structures of the cold-induced and heat-induced D states are similar. Both states exhibit similar (1)H-(15)N HSQC spectra and the same pattern of peptidyl-prolyl peptide bond configurations by NMR, and both appear to be similarly expanded compared with the S state based on analysis of SAXS data. Whereas in other proteins the cold-denatured states have been found to be slightly more compact than the heat-denatured states, these two states occupy similar volumes in IscU.

Figure 1

Energetic landscape of IscU. (A) IscU exists in equilibrium between a structured (S) state (PDB ID: 24LX) (8) and a disordered (D) state. (B) Temperature dependence of the fraction of the protein molecules in the S state, ([S]/([S]+[D])), as determined from the relative intensities of NMR signals assigned to W76 (black circles) and K128 (red circles) in the two conformational states. The curves resulted from fitting each data set to the Gibbs-Helmholtz equation (Table 1). (C) DSC thermograms of IscU (black) and D39A IscU (red).

Figure 2

¹H-¹⁵N HSQC spectra of IscU collected at the temperatures indicated, showing the collapse of the spectrum upon cooling or warming. Boxes highlight the resonances used to determine the S- and D-state populations for thermodynamic analysis (Fig. 1B).

Figure 3

Temperature-dependent SAXS studies of monomeric IscU prepared with 10 mM TCEP in an anaerobic chamber. (A) R_g values calculated from SAXS data collected at different temperatures. (B) Molecular mass of IscU as determined from SAXS data by the I(0) (black) and V_c (red) methods. The lines indicate the molecular masses of monomeric (13.8 kDa) and dimeric (27.7 kDa) IscU.

Figure 4

Low-resolution structural analysis of IscU by SAXS. (A) D-state R_g values calculated from experimental SAXS data using Eq. 1 (black) and from disordered structures selected from a two-state MES (red). (B) R_g values for members of the ensemble of structures used for the MES analysis. (C) Fractional S-state population derived from the conformers selected by the two-state MES fit (red) compared with that determined by NMR (black).

Figure 5

MES results for temperature-dependent IscU SAXS data. (A) Experimental SAXS data (circles) collected at different temperatures are compared with fits to MES against one structure (blue) and two structures (red). $\mathcal{X}$ and ${\mathcal{X}}_{\text{free}}$ statistics are shown for the two-structure MES fits. For reference, the fit (green) to the single structure (R_g of 22 Å) selected for IscU at 25°C is shown for 1°C, 5°C, and 45°C. (B) Structures selected in the two-state MES fits to the experimental SAXS data at different temperatures.