NMR SPECTROSCOPY OF IRON-SULFUR PROTEINS

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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.