Inequivalent contribution of the five tryptophan residues in the C-lobe of human serum transferrin to the fluorescence increase when iron is released.
Human serum transferrin (hTF), with two Fe3+ binding lobes, transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells, and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant time scale (2-3 min), the factors critical to iron release include pH, anions, a chelator, and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (+/-sTFR). Only four of the five recombinant Trp --> Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: k(obsC1) reports conformational changes in the C-lobe initiated by the TFR, and k(obsC2) is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.
James
Nicholas G
NG
Department of Biochemistry, College of Medicine, University of Vermont, 89 Beaumont Avenue, Burlington 05405, Vermont, USA.
Byrne
Shaina L
SL
Steere
Ashley N
AN
Smith
Valerie C
VC
MacGillivray
Ross T A
RT
Mason
Anne B
AB
D000042:Absorption; D018360:Crystallography, X-Ray; D005453:Fluorescence; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D017354:Point Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D011990:Receptors, Transferrin / Q000378:metabolism; D044967:Serum; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D011990:Receptors, Transferrin; D014168:Transferrin; D014364:Tryptophan; D007501:Iron