Qing-Yu He

Identification of platination sites on human serum transferrin using (13)C and (15)N NMR spectroscopy.

Abstractu2002Reactions between various apo and metal-bound forms of human serum transferrin (80u2009kDa) and the recombinant N-lobe (40u2009kDa) with [Pt(en)Cl2] or cis-[PtCl2(NH3)2] have been investigated in solution via observation of [1H,15N] NMR resonances of the Pt complexes, [1H,13C] resonances of the eCH3 groups of the protein methionine residues, and by chromatographic analysis of single-site methionine mutants. For the whole protein, the preferred Pt binding site appears to be Met256. Additional binding occurs at the other surface-exposed methionine (Met499), which is platinated at a slower rate than Met256. In contrast, binding of similar Pt compounds to the N-lobe of the protein occurs at Met313, rather than Met256. Met313 is buried in the interlobe contact region of intact transferrin. After loss of one chloride ligand from Pt and binding to methionine sulfur of the N-lobe, chelate-ring closure appears to occur with binding to a deprotonated backbone amide nitrogen, and the loss of the other chloride ligand. Such chelate-ring closure was not observed during reactions of the whole protein, even after several days.

The chloride effect is related to anion binding in determining the rate of iron release from the human transferrin N-lobe.

The major function of human transferrin is to deliver iron from the bloodstream to actively dividing cells. Upon iron release, the protein changes its conformation from closed to open. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factors, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodworth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an interdependent manner: chloride retards iron release at neutral pH, but accelerates the reaction at acidic pH. The present study supports this idea and further details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the apoprotein. The negative effect seems to originate from competition between chloride and the chelator for an anion-binding site(s) near the metal centre. With decreasing pH, the strength of anion binding to hTF/2N increases linearly, decreasing the contribution of competition with the chelator. In the meantime, the open or loose conformation of hTF/2N, induced by the protonation of critical residues such as the Lys-206/Lys-296 pair at low pH, enables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by the chelator, leading to a positive anion effect. Disabling one or more of the primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, substantially decreases the anion-binding ability of the resulting mutant proteins. In these cases, the competition for the remaining binding residue(s) is increased, leading to a negative chloride effect or, at most, a very small positive effect, even at low pH.

Anion exchange in human serum transferrin N-lobe: a model study with variant His249Ala

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. Anion exchange occurs by two pathways at pHxa07.4 and 25xa0°C: an NTA-independent dissociative pathway to form a carbonate-free intermediate Fe-H249A (Eq.xa01) that subsequently reacts with NTA (Eq.xa02):1% MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr % 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrpipeea0xe9Lq-Jc9 % vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x % fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeOraiaabw % gacqGHsislcaqGibGaaeOmaiaabsdacaqG5aGaaeyqaiabgkHiTiaa % boeacaqGpbWaaSbaaSqaaiaaiodaaeqaaOGaaGjbVlaaysW7daGdKa % WcbaGaam4AamaaBaaameaacaaIXaGaaiilaiaaicdaaeqaaSGaeyyp % a0JaaGimaiaac6cacaaIYaGaaGOnaiaaiAdacaaMe8Uaae4CamaaCa % aameqabaGaeyOeI0IaaGymaaaaaSqabOGaayPKHaGaaGjbVlaaysW7 % caqGgbGaaeyzaiabgkHiTiaabIeacaqGYaGaaeinaiaabMdacaqGbb % Gaey4kaSIaaeisaiaaboeacaqGpbWaaSbaaSqaaiaaiodaaeqaaOWa % aWbaaSqabeaacqGHsislaaaaaa!5F93!

Expression, purification, and characterization of recombinant nonglycosylated human serum transferrin containing a C-terminal hexahistidine tag.

{rm Fe} hbox{-} {rm H249A}hbox{-} {rm CO}_3 ;;buildrel {k_{1,0} = 0.266;{rm s}^{ - 1} } over longrightarrow ;;{rm Fe} hbox{-} {rm H249A} + {rm HCO}_3 ^ -

Mutagenesis of the aspartic acid ligands in human serum transferrin: lobe-lobe interaction and conformation as revealed by antibody, receptor-binding and iron-release studies

2% MathType!MTEF!2!1!+- % feaaeaart1ev0aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn % hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr % 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrpipeea0xe9Lq-Jc9 % vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x % fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeOraiaabw % gacqGHsislcaqGibGaaeOmaiaabsdacaqG5aGaaeyqaiabgUcaRiaa % bIeacaqGobGaaeivaiaabgeadaahaaWcbeqaaiaabkdacqGHsislaa % GccaaMe8UaaGjbVpaaoqcaleaacaWGRbWaaSbaaWqaaiaaikdaaeqa % aSGaeyypa0JaaGOnaiaaikdacaGGUaGaaGimaiaaysW7caqGnbWaaW % baaWqabeaacqGHsislcaaIXaaaaSGaaGjbVlaabohadaahaaadbeqa % aiabgkHiTiaaigdaaaaaleqakiaawkziaiaaysW7caaMe8UaaeOrai % aabwgacqGHsislcaqGibGaaeOmaiaabsdacaqG5aGaaeyqaiabgkHi % Tiaab6eacaqGubGaaeyqaaaa!6229!

SPECTROPHOTOMETRIC TITRATION WITH COBALT(III) FOR THE DETERMINATION OF ACCURATE ABSORPTION COEFFICIENTS OF TRANSFERRINS

{rm Fe} hbox{-} {rm H249A} + {rm HNTA}^{{rm 2} - } ;;buildrel {k_2 = 62.0;{rm M}^{ - 1} ;{rm s}^{ - 1} } over longrightarrow ;;{rm Fe} hbox{-} {rm H249A} hbox{-} {rm NTA}