In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys3...

Chloroplast fructose 1,6-bisphosphatase (FBPase) is activated by reduction of a regulatory disulfide through thioredoxin f (Trx f). In the course of this reduction a transient mixed disulfide is formed linking covalently Trx f with FBPase, which possesses three Cys on a loop structure, two of them forming the redox-active disulfide bridge. The goal of this study was to identify the Cys involved...

A method for determining the kinetic fate of structured disulfide species (i.e., whether they are preferentially oxidized or reshuffle back to an unstructured disulfide species) is introduced. The method relies on the sensitivity of unstructured disulfide species to low concentrations of reducing agents. Because a structured des species that preferentially reshuffles generally first rearranges ...

—The disulfide bonds in γ-46 gliadin were identified: Cys 173 –Cys 192 , Cys 212 –Cys 291 , Cys 165 –Cys 199 (or Cys 200 ), Cys 283 –Cys 200 (or Cys 199 ). The disulfide-containing peptides were obtained by limited hydrolysis of the intact protein with chymotrypsin at an enzyme/substrate ratio of 1:1000 at 20°C for 22 h with subsequent digestion of disulfide-containing fragments with trypsin an...

A novel immobilized N-chlorosuccinimide resin was developed for peptide disulfide bond formation in combinatorial libraries. The resin is prepared in a simple two-step process from commercial starting materials. Disulfide formation is initiated by adding a peptide solution to the resin, and excess reagent is removed by a convenient filtration upon completion of disulfide formation. Completion o...

Durand, Kirt Lenroy. Ph.D., Purdue University, December 2014. Determination of Disulfide Bond Connecting Patterns via Tandem Mass Spectrometry (MS) and Biomolecular Ion/Radical Reactions. Major Professor: Yu Xia. Disulfide bond formation is one of the most common post translational modifications to occur in proteins and naturally occurring peptides. Disulfide bond formation plays a critical rol...

A liquid chromatography tandem-mass spectrometry method was developed to map the eleven disulfide bonds in Pfs25, a malaria transmission-blocking vaccine candidate. The compact and complex nature of Pfs25 has led to difficulties in prior peptide mapping efforts. Here, we report confirmation of proper disulfide pairing of a recombinant Pfs25, by optimizing denaturation and digestion with trypsin...

In this issue of Cell, show that there is a disulfide relay system in the intermembrane space (IMS) of mitochondria that is comprised of the proteins Mia40 and Erv1. This disulfide relay system promotes the import and oxidative folding of proteins. Oxidized Mia40 traps newly imported proteins through mixed disulfide bridges. Subsequent isomerization of these disulfide bridges allows the importe...

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end...

Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant...