Intrinsic fluorescence reports a global conformational change in the N-lobe of human serum transferrin following iron release.
Transferrins have been extensively studied in order to understand how they reversibly bind and release iron. Human serum transferrin (hTF) is a single polypeptide chain that folds into two lobes (N- and C-lobe); each lobe binds a single ferric ion. Iron release induces a large conformational change in each lobe. At the putative endosomal pH of 5.6, measurement of the increase in intrinsic fluorescence upon iron release from the recombinant N-lobe yields two rate constants: 8.9 min-1 and 1.3 min-1. Direct monitoring of iron release from the N-lobe at pH 5.6 (by the decrease in absorbance at 470 nm) gives a single rate constant of 9.1 min-1, definitively establishing that the faster rate constant in the fluorescent studies is due to iron release. To further elucidate the molecular basis of the intrinsic fluorescence change (and the source of the slower rate constant), we examined the contributions of the three individual tryptophan residues in the N-lobe (Trp8, Trp128, and Trp264). Three double mutants, each containing the single remaining tryptophan residue, were produced. In the iron-bound N-lobe, Trp128 and Trp264 are quenched by iron and account for almost the entire fluorescent signal when iron is released. As for the wild-type N-lobe, the fluorescence increase for each of these mutants is best fit by a double-exponential function indicating two processes. Trp8 is severely quenched under all conditions, making virtually no contribution to the signal. Additionally, a mutant lacking all three Trp residues allows assignment of the fluorescent signal completely to the three tryptophan residues and observation of the presence of one (or more) tyrosinates in the N-lobe that have physiological significance in the uptake of iron.
James
Nicholas G
NG
Department of Biochemistry, University of Vermont, Burlington, Vermont 05405, USA.
Berger
Christopher L
CL
Byrne
Shaina L
SL
Smith
Valerie C
VC
MacGillivray
Ross T A
RT
Mason
Anne B
AB
D000042:Absorption; D018360:Crystallography, X-Ray; D003553:Cystine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan
D050505:Mutant Proteins; D014168:Transferrin; D003553:Cystine; D014364:Tryptophan; D007501:Iron