Ferritin is a multisubunit protein that concentrates and sequesters as many as 4,500 iron atoms within a hollow protein shell in animal, plant and bacterial cells. There are at least three ferritin genes in animals that differ in expression in different tissues, under different environment conditions and with development; their rates and mechanisms of iron accumulation, storage and turnover are also different. Our goal is to investigate the mechanism of action of ferritin and the structural basis of these functional differences by using X-ray crystallography to determine and compare the three-dimensional structures of recombinant amphibian red H and L ferritins and a set of single site mutants with altered function under different experimental conditions. Particular emphasis will be placed on mutants that eliminate or restore the ability to form the early iron-tyrosine intermediate that forms only in H- ferritin, mutations involving a set of mobile sidechains associated with a spine of hydration that we have identified in previous studies and mutations near the symmetry axes. We will also use a package developed in this laboratory specifically for modeling large macromolecular assemblies to model and compare electrostatic effects in the various structures. The functional properties of the various proteins will be determined and compared in solution, principally by EXAFS, resonance Raman, Mossbauer and uv-visible absorption spectroscopy. The basis of this proposal is our earlier studies of wild type amphibian red cell L-ferritin and two mutants which indicated that the ferritin structure is more plastic than had previously been appreciated and can be differentially solvated. We wish to elucidate the rules that govern these structural changes and their relationship to function, particularly the differences in L- and H- ferritins. Since the subunits of ferritin are four-helix bundles, this study will provide insight into how this common structural motif in proteins accommodates to changes in sequence and external conditions. The mechanism of action of ferritin is relevant to understanding the molecular basis of iron metabolism in normal health and in diseases such as thalassemia, hemochromatosis and sickle cell anemia or other conditions requiring hypertransfusion or hemodialysis.
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