Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C-lobe of ovotransferrin.
The interdomain disulfide bond present in the C-lobe of all the transferrins was postulated to restrict the domain movement resulting in the slow rate of iron uptake and release. In the present study, the conformational stability and iron binding properties of a derivative of the isolated C-lobe of ovotransferrin in which the interdomain disulfide bond, Cys478-Cys671 was selectively reduced and alkylated with iodoacetamide were compared with the disulfide intact form at the endosomal pH of 5.6. Pyrophosphate and chloride mediated iron release kinetics showed no difference between the disulfide-intact and disulfide-reduced/alkylated forms; the two protein forms yielded similar observed rate constants showing an apparent hyperbolic dependency for anion concentrations. The conformational stability evaluated by unfolding and refolding experiments was greater for the disulfide-intact form than for the disulfide-reduced/alkylated form: the deltaG(D)H2O values at 30 degrees C obtained by using urea were 9.0+/-0.8 and 6.0+/-0.4 kJ/mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2+/-0.9 and 4.3+/-0.5 kJ/mol. The dissociation constant of iron (kd) was almost the same for the two protein forms, and it varied only subtly with urea concentrations but increased markedly with GdnHCl concentrations. The nonidentical values of deltaG(D)H2O and kd for urea and GdnHCl can be attributed to the ionic nature of the later denaturant, in which chloride anion may influence the structure and iron uptake-release properties of the ovotransferrin C-lobe. Taken together, we conclude that the interdomain disulfide bond has no effect on the iron uptake and release function but significantly decreases the conformational stability in the C-lobe.
Muralidhara
B K
BK
The Research Institute for Food Science, Kyoto University, Uji, Japan.
Hirose
M
M
D003207:Conalbumin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000032:analysis; D011756:Diphosphates / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D014508:Urea / Q000737:chemistry
D011756:Diphosphates; D004220:Disulfides; D010455:Peptides; D003207:Conalbumin; D014508:Urea; D007501:Iron; D019791:Guanidine; D003545:Cysteine