The Complex Energy Landscape of the Protein IscU.
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.
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin; National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin.
Jameson R
JR
Bothe
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin.
Marco
M
Tonelli
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin.
Ibrahim K
IK
Ali
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin; National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin.
Ziqi
Z
Dai
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin; National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin.
Ronnie O
RO
Frederick
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin.
William M
WM
Westler
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin; National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, Wisconsin. Electronic address: jmarkley@wisc.edu.
John L
JL
Markley
D003080:Cold Temperature; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D055550:Protein Stability; D013455:Sulfur / Q000378:metabolism; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D013455:Sulfur; D007501:Iron