Virtually all living organism require iron. The properties of iron in aqueous solution imply that organisms dependent upon iron are also dependent on specific iron- binding molecules to maintain the essential element in soluble, bioavailable and non-toxic form. In all vertebrates, as well as in many lower organisms, the functions of iron transport, storage and detoxification are managed by specialized proteins of iron metabolism, the transferrins and ferritins. Human transferrin, the major interest of this research, serves in the circulatory transport of iron for cellular needs. The protein experiences 100-200 cycles of iron transport during its lifetime, so that understanding its biological activity demands understanding its mechanisms of iron binding and release. For most cells, uptake of iron from transferrin is a receptor- mediated event, with release rates of iron from transferrin modulated by binding of the protein to its receptor. The primary focus of this research, therefore, is with identifying the receptor-recognition locus of transferrin, and discerning how the transferrin receptor regulates release of iron. Since iron release is also sensitive to binding of simple anions by transferrin, this work also investigates the kinetically active sites on transferrin which respond to these anions. Toward these ends the PI will employ, in concert, the methods and concepts of physical biochemistry and cell biology, along with site- directed mutagenesis guided by transferrin crystallography. Control studies, using circular dichroism and 15N NMR spectroscopies, competition binding with synthetic peptides modeling candidate receptor- binding sites, and crystallography of promising mutants, will be undertaken to ensure that mutagenesis does not induce remote changes responsible for observed receptor-binding and kinetic alterations. A related concern centers on the magnetic properties of ferritin, how these properties evolve with size of the protein's polynuclear iron core, and how they affect magnetic resonance imaging (MRI). Understanding the fundamental chemistry of iron transport, storage and utilization will provide the rational basis for managing the multitude of diseases where aberrations of iron metabolism contribute to cellular injury and death.
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