Organisms dependent upon iron also require specific iron-binding molecules to maintain the essential element in soluble, bioavailable and non-toxic form. In vertebrates, and in many organisms lower on the phylogenetic tree, the functions, of iron transport, storage and detoxification are managed by specialized proteins of iron metabolism, transferrin and ferritin. Proposed studies of transferrin and transferrin-receptor interactions will explore the molecular mechanism regulating the receptor-mediated delivery of iron to cells by transferrin, and the role of the transferrin receptor in modulating release of iron from transferrin to the iron-dependent cell. A particular goal is to localize the receptor recognition site(s) on the transferrin molecule. In pursuit of these aims, use will be made of site-mutagenized recombinant transferrin. Site-directed mutagenesis will be guided by insights gleaned from protein crystallography, kinetic analyses of iron release from transferrin and its complexes with the transferrin receptor, and cellular studies of iron uptake from transferrin and single-site transferrin fragment. Investigations of newly described and cloned insect transferrin will further extend our understanding of how transferrin came to be and how the functions displayed by the protein of today evolved. A related interest centers on the magnetic properties of the polynuclear iron core, and iron on its way to the core, of ferritin. Neither the susceptibility of ferritin as a function of core size, nor the effect of this susceptibility on magnetic relaxation times of solvent water protons (an important parameter in the clinical assessment of iron overload by magnetic resonance imaging), is understood. Accordingly, magnetic interactions among the iron atoms of the ferritin core will be analyzed, with a focus on whether these change as core size changes, and how they influence relaxation times of solvent water protons. The information sought will be obtained by static magnetic susceptibility measurements with a SQUID-based magnetometer, correlated with magnetic relaxation rate studies over a frequency range of interest in magnetic resonance imaging. A final concern is with uteroferrin, a prototype of the purple acid phosphatases with binuclear iron centers. Of particular interest is how inhibitory oxoanions bind to the metal center, and whether one or both metal ions are involved in such binding. This problem will be addressed by ENDOR and multifrequency ESEEM spectroscopy of native and mixed-metal enzymes complexed with competitive and noncompetitive oxoanion inhibitors.
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