The Complex Energy Landscape of the Protein IscU

Ibrahim K Ali,William M Westler,John L Markley,Jameson R Bothe,Ronnie O Frederick,Marco Tonelli,Ziqi Dai

doi:10.1016/j.bpj.2015.07.045

Ibrahim K Ali, William M Westler + Show 5 more

Open Access

https://doi.org/10.1016/j.bpj.2015.07.045

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Journal: Biophysical Journal	Publication Date: Sep 1, 2015
Citations: 14	License type: cc-by

Affiliation: University of Wisconsin–Madison

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 1H-15N 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.

Highlights

Cold denaturation is a fundamental aspect of the protein free-energy landscape, general questions remain regarding its associated structures and energetics [1]
The generally accepted model of cold denaturation involves a reduction of the hydrophobic effect such that the hydration of nonpolar groups becomes more favorable at low temperatures
The D state yields a poorly dispersed 1H-15N heteronuclear single quantum coherence (HSQC) spectrum indicative of dynamic disorder (Fig. 2), and the lack of secondary chemical shifts indicates that the D state contains minimal secondary structure [34]

Summary

Introduction

Cold denaturation is a fundamental aspect of the protein free-energy landscape, general questions remain regarding its associated structures and energetics [1]. The biggest challenge in answering basic thermodynamic questions about cold denaturation is identifying proteins that undergo the process without the assistance of destabilizing effects such as the presence of alcohols or denaturants, confinement in micelles, or extreme pressure [1,2,3,4,5,6,7,8,9,10] These destabilizing effects have been crucial in allowing insights into cold-denatured states, it is difficult to fully decouple their effect on the protein-folding process from both an energetic and a structural standpoint. This is a key issue because hydration and the hydrophobic

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Conclusion