Iron-sulfur proteins are ubiquitous in nature and essential to life. Members of this class of metalloprotein play critical roles in biochemical processes ranging from respiration and detoxification to gene regulation and chemical sensing. Defects in iron-sulfur proteins are linked to a large number of human diseases. The electronic properties of iron-sulfur proteins, which impact on their function, are modulated by the geometry of their iron centers and by interactions between electrons on the metal(s) and the ligands provided by the protein. Our overall goals are to determine how protein-protein interactions facilitate iron-sulfur cluster assembly and modulate electron-nuclear interactions at the active sites of iron-sulfur proteins so as to facilitate electron transfers. Our experimental approach to understanding sequence-structure-function relationships in iron-sulfur proteins combines NMR spectroscopy and quantum chemical calculations with information from X-ray crystallography. NMR spectra contain exquisitely sensitive information about electron-nuclear interactions. This information, which is unavailable from X-ray crystal structures, provides insights into the chemical properties of the iron centers, details of their geometry, strengths of hydrogen bonds, proton affinities, and patterns of electron delocalization. NMR thus serves as a window for viewing the properties of the cluster that control redox potentials and regulate pathways of electron transfer and redox dependent changes in pKa values. In the past, we have applied this approach to investigations of single iron-containing proteins and proteins that interact with them. We propose here to extend these investigations to biologically important protein-protein complexes that involve iron centers.
Our specific aims are: (1) to determine the mechanism of coupling between proton and electron transfer in the Rieske protein from Thermus thermophilus;(2) to determine how protein:protein complexes affect electron-nuclear interactions and to determine mechanisms of electron transfer in three model biological systems (rubredoxin:FNR, stearoyl-acyl carrier protein desaturase, and toluene monooxygenase system);(3) to determine mechanisms of chaperone-assisted iron-sulfur cluster assembly in Escherichia coli. To achieve these aims, we will make use of state-of-the-art approaches to protein production and isotopic labeling, NMR data collection and analysis, modeling of structures and complexes, and quantum chemical calculations.
|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|
|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|
|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|
|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; 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|
|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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