Iron sequestration in blood plasma by transferrin and delivery of this iron via receptor mediated endocytosis to various tissues, such as bone marrow and liver, are biologically essential functions. This natural physiological process is also utilized in the supply of iron to neoplastic tissues. The fundamentals of structure-function relationships in transferrin are essential to understanding the molecular basis, of diseases that result in defects in iron delivery and for the rational design of interventions in such diseases. This proposal involves elucidation of the structure-function relationships in recombinant human serum transferrin and hen ovotransferrin derived from a series of site-directed mutants. Metal binding affinity is measured by determination of kinetic constants for release of the bound metal ion to a competing, high affinity chelator as a function of pH and ionic strength. Binding affinities will be correlated to the conformational state of the protein. Continuing X-ray crystallographic studies of selected mutants will determine the precise structural rearrangements, which account for the functional differences observed in the mutated proteins. Solving the structure of recombinant, non-glycosylated human serum transferrin will be a particular focus, because this construct will be used to study site-directed mutants in the C-lobe. Another major aim will be to continue efforts to establish reliable indices of the """"""""open"""""""" and """"""""closed"""""""" conformations of each lobe in solution as a function of pH, ionic strength, and degree of metal ligation. Various physicochemical approaches, such as EPR of spin labeled transferrin, fluorescence resonance energy transfer, fluorescence spectroscopy and in some cases, NMR, will be used to accomplish this aim. The conformational dependence in the recognition of various transferrins and mutants of transferrin by the recombinant soluble transferrin receptor will be assessed by techniques, such as analytical ultracentrifugation, fluorescence resonance energy transfer and EPR of spin labeled proteins. The rate of iron removal from transferrin and transferrin mutants will be measured in the presence of the receptor. In addition, binding studies of transferrin and mutated transferrins to receptors on living cells will provide measurements of both binding affinity and ability to deliver iron to the cells and will indicate the physiological relevance of the in vitro work.
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