The methods of NMR spectroscopy and computational chemistry will be used to investigate structure-function relationships in iron- sulfur proteins of biological and biomedical importance. Proteins to be studied include those that contain single metal sites with 4 Cys, 3 Cys/1 His, and 2 Cys/2His ligation (rubredoxins and zinc/iron fingers), various classes of [2Fe-2S] 4 Cys clusters (plant, bacterial, and vertebrate ferredoxins), and two classes of [2Fe-2S] 2 Cys/2 His clusters (Rieske proteins). The roles of various interactions (metal coordination to particular amino acid side chains, hydrogen bonds, and nearby charges) will be evaluated by selective protein modifications--by mutagenesis and chemical synthesis or by incorporating different metal ions. A unique feature of our approach is our demonstrated ability to combine information from paramagnetic NMR spectra, x-ray crystallography, and quantum mechanical calculations to evaluate structural models for metal centers in proteins. Information of this kind is of fundamental importance for the design of drugs that target metal binding sites. It is expected that this approach will yield new insights about the role that various interactions play in stabilizing the protein in its accessible oxidation states. The growing database of assigned hyperfine 1H, H, 13C, and 15N chemical shifts for iron-sulfur proteins should be useful for determining cluster categories in newly discovered proteins from NMR data and for developing amino acid consensus sequences associated with particular cluster types. Novel approaches promise to improve the quality of NMR structures of paramagnetic proteins derived from NMR data. The usual constraints derived from NOEs and three-bond J-couplings from the diamagnetic region will be supplemented by information from field-dependent one-bond dipolar couplings arising from partial ordering of the proteins by the magnetic field, constraints derived from analysis of hyperfine-shifted resonances, and artificial potentials derived from the database of known protein structures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058667-02
Application #
6151238
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1999-02-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$183,362
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Dai, Ziqi; Kim, Jin Hae; Tonelli, Marco et al. (2014) pH-induced conformational change of IscU at low pH correlates with protonation/deprotonation of two conserved histidine residues. Biochemistry 53:5290-7
Kim, Jin Hae; Bothe, Jameson R; Frederick, Ronnie O et al. (2014) Role of IscX in iron-sulfur cluster biogenesis in Escherichia coli. J Am Chem Soc 136:7933-42
Markley, John L; Kim, Jin Hae; Dai, Ziqi et al. (2013) Metamorphic protein IscU alternates conformations in the course of its role as the scaffold protein for iron-sulfur cluster biosynthesis and delivery. FEBS Lett 587:1172-9
Cai, Kai; Frederick, Ronnie O; Kim, Jin Hae et al. (2013) Human mitochondrial chaperone (mtHSP70) and cysteine desulfurase (NFS1) bind preferentially to the disordered conformation, whereas co-chaperone (HSC20) binds to the structured conformation of the iron-sulfur cluster scaffold protein (ISCU). J Biol Chem 288:28755-70
Hsueh, Kuang-Lung; Tonelli, Marco; Cai, Kai et al. (2013) Electron transfer mechanism of the Rieske protein from Thermus thermophilus from solution nuclear magnetic resonance investigations. Biochemistry 52:2862-73
Kim, Jin Hae; Tonelli, Marco; Frederick, Ronnie O et al. (2012) Specialized Hsp70 chaperone (HscA) binds preferentially to the disordered form, whereas J-protein (HscB) binds preferentially to the structured form of the iron-sulfur cluster scaffold protein (IscU). J Biol Chem 287:31406-13
Fleischhacker, Angela S; Stubna, Audria; Hsueh, Kuang-Lung et al. (2012) Characterization of the [2Fe-2S] cluster of Escherichia coli transcription factor IscR. Biochemistry 51:4453-62
Hansen, D Flemming; Westler, William M; Kunze, Micha B A et al. (2012) Accurate structure and dynamics of the metal-site of paramagnetic metalloproteins from NMR parameters using natural bond orbitals. J Am Chem Soc 134:4670-82
Kim, Jin Hae; Tonelli, Marco; Markley, John L (2012) Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase. Proc Natl Acad Sci U S A 109:454-9
Dai, Ziqi; Tonelli, Marco; Markley, John L (2012) Metamorphic protein IscU changes conformation by cis-trans isomerizations of two peptidyl-prolyl peptide bonds. Biochemistry 51:9595-602

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