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PMID:15323571

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Citation

Okamoto, I, Mizutani, K and Hirose, M (2004) Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants. Biochemistry 43:11118-25

Abstract

Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).

Links

PubMed Online version:10.1021/bi049147j

Keywords

Amino Acid Sequence/genetics; Animals; Binding, Competitive/genetics; Calorimetry, Differential Scanning; Chickens; Conalbumin/chemistry; Conalbumin/genetics; Ferric Compounds/chemistry; Ferric Compounds/metabolism; Iron-Binding Proteins/chemistry; Iron-Binding Proteins/genetics; Ligands; Models, Chemical; Mutagenesis, Site-Directed; Peptide Fragments/chemistry; Peptide Fragments/genetics; Phenylalanine/genetics; Pichia/genetics; Protein Binding/genetics; Protein Denaturation; Thermodynamics; Tyrosine/genetics

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CHICK:TRFE

located_in

GO:0005615: extracellular space

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TRFE

enables

GO:0008199: ferric iron binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

CHICK:TRFE

part_of

GO:0005615: extracellular space

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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