Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site.
The effects of single amino acid substitution on the thermal stability of the N-terminal half-molecule of human transferrin and its iron-binding affinity have been studied by high-sensitivity scanning calorimetry. All site-directed mutations are located on the surface of the binding cleft, and they are D63-->S, D63-->C, G65-->R, H207-->E and K206-->Q. Differential scanning calorimetry results show that the mutations do not significantly alter the conformational stability of the apo-forms of the proteins. The changes in free energy of unfolding relative to the wild-type protein range from 0.83 to -2.4 kJ/mol. The D63-->S, G54-->R and H207-->E mutations slightly destabilize the apo-protein, while the D63-->C and K206-->Q mutations increase its stability by a small amount. However, there are large compensating enthalpy-entropy changes caused by all mutations. All mutants bind ferric ion, but with different affinities. Replacement of Asp-63 by either Ser or Cys decreases the apparent binding constant by 5-6 orders of magnitude. The G65-->R mutation also decreases the apparent binding constant by 5 orders of magnitude. The K206-->Q mutation increases the apparent binding constant by 20-fold, while the H207-->E mutation does not significantly change the apparent iron-binding affinity of the half-molecule.
Lin
L N
LN
Department of Chemistry, University of Massachusetts, Amherst 01003.
Mason
A B
AB
Woodworth
R C
RC
Brandts
J F
JF
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D006801:Humans; D007501:Iron / Q000378:metabolism*; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014168:Transferrin; D007501:Iron