Structural and functional consequences of removal of the interdomain disulfide bridge from the isolated C‐lobe of ovotransferrin

Abstract

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-reduced0alkylated 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-reduced0alkylated form: the ⌬G D H 2 O values at 30 8C obtained by using urea were 9.0 6 0.8 and 6.0 6 0.4 kJ0mol for the former and latter protein forms, respectively, and the corresponding values obtained by using guanidine hydrochloride were 6.2 6 0.9 and 4.3 6 0.5 kJ0mol. The dissociation constant of iron~k d ! 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 ⌬G D H 2 O and k d 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.

Antiangiogenic Activity of a Domain Deletion Mutant of Tissue Plasminogen Activator Containing Kringle 2

“…Other studies have shown that selective reduction of disulphide bonds is possible with limited use of DTT. 24 We found that 1 mmol/L DTT was sufficient to selectively reduce the Cys 264 -Cys 395 disulphide bond without compromising the conformational state of the kringle 2 module, as evidenced by the fact that K2 was still able to bind lysine-Sepharose after DTT treatment. Human and mouse endothelial cell migration was significantly reduced in a scratch wound assay after treatment with K2 containing tPA fragments, as was in vivo angiogenesis in a mouse sponge angiogenesis assay.…”

Comparative Quantitative Phosphoproteomic Analysis of the Chicken Egg during Incubation Based on Tandem Mass Tag Labeling

“…OTf has a broad-spectrum antibacterial ability, mainly due to its binding capability to the iron ions which is necessary for microbial growth. 49 It has been confirmed that phosphorylated OTf significantly enhanced antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. 50 Thus, we can speculate that the increased phosphorylation of Tyr 191 might promote the antibacterial ability of egg white and yolk, which in turn protect chicken embryo from bacterial infections.…”

The Folding Process of Acylphosphatase from Escherichia coli is Remarkably Accelerated by the Presence of a Disulfide Bond

“…26 Except for a few cases, 39 it has been generally reported that disulfide bonds are responsible for a stabilization of the overall three-dimensional structure of proteins of different sizes, regardless of their structural organization. [40][41][42][43][44][45][46][47] It is clear that the native disulfide bond of EcoAcP also has a marked influence on the folding process of this protein. EcoAcP exhibits two-state refolding across the whole range of denaturant concentrations studied, with no early folding event within the dead time of the instrument (Fig.…”