We propose to attempt to identify the amino acid sidechains of the specific iron and anion binding sites of the proteins, transferrin and ovotransferrin (conalbumin). Furthermore, we seek to identify those amino acids which serve as ligands to the iron and anions and to determine the spatial disposition of the binding site amino acids. A major investigational tool will be high resolution nuclear magnetic resonance. In order to simplify interpretation of the spectral data, N- and C-terminal """"""""half-molecules"""""""" of the transferrins, each with its attendant binding site will be studied. In the case of ovotransferrin, protein with selectively deuterated 15N- or F-enriched amino acids will be produced by feeding birds a defined diet containing the modified amino acids. Alternatively we will seek to express the transferrin genes in microorganisms in order to carry out more efficiently the incorporation of amino acids enriched with NMR observable or silent nuclei. Point mutations in the gene will aim at modifying iron and anion binding behaviors on the transferrin half-molecules. These substitutions will substantially simplify the nmr spectra and therefore their interpretation. Differences in the binding sites will be sought. Peptide mapping studies will seek to locate the various ligands in the primary amino acid sequence. Mossbauer studies of 57Fe-transferrins will be aimed at understanding the environment of the iron-binding sites.
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