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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK021739-21
Application #
6649859
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Badman, David G
Project Start
1978-12-01
Project End
2006-04-30
Budget Start
2003-07-01
Budget End
2004-04-30
Support Year
21
Fiscal Year
2003
Total Cost
$311,648
Indirect Cost
Name
University of Vermont & St Agric College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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Das, Anupam; Nag, Sagarika; Mason, Anne B et al. (2016) Endosome-mitochondria interactions are modulated by iron release from transferrin. J Cell Biol 214:831-45
Mathies, Guinevere; Gast, Peter; Chasteen, N Dennis et al. (2015) Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz. J Biol Inorg Chem 20:487-96
Luck, Ashley N; Mason, Anne B (2013) Structure and dynamics of drug carriers and their interaction with cellular receptors: focus on serum transferrin. Adv Drug Deliv Rev 65:1012-9
Deblonde, Gauthier J-P; Sturzbecher-Hoehne, Manuel; Mason, Anne B et al. (2013) Receptor recognition of transferrin bound to lanthanides and actinides: a discriminating step in cellular acquisition of f-block metals. Metallomics 5:619-26
Sturzbecher-Hoehne, Manuel; Goujon, Christophe; Deblonde, Gauthier J-P et al. (2013) Sensitizing curium luminescence through an antenna protein to investigate biological actinide transport mechanisms. J Am Chem Soc 135:2676-83
Luck, Ashley N; Bobst, Cedric E; Kaltashov, Igor A et al. (2013) Human serum transferrin: is there a link among autism, high oxalate levels, and iron deficiency anemia? Biochemistry 52:8333-41
Steere, Ashley N; Chasteen, N Dennis; Miller, Brendan F et al. (2012) Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin. Biochemistry 51:2113-21
Steere, Ashley N; Byrne, Shaina L; Chasteen, N Dennis et al. (2012) Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH. Biochim Biophys Acta 1820:326-33
Luck, Ashley N; Mason, Anne B (2012) Transferrin-mediated cellular iron delivery. Curr Top Membr 69:3-35

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