The biotin switch assay for detection of protein S-nitrosation has been widely used in the field of nitric oxide and redox signaling. However, here we found that there is experimental and theoretical interference of intermolecular disulfide bonds in S-nitrosated protein identification with avidin purification after biotin switch method: proteins linked to S-nitrosated proteins by intermolecular...

—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...

Disulfide bonds are primary covalent cross-links formed between two cysteine residues in the same or different protein polypeptide chains, which play important roles in the folding and stability of proteins. However, computational prediction of disulfide connectivity directly from protein primary sequences is challenging due to the nonlocal nature of disulfide bonds in the context of sequences,...

UNLABELLED Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human...

Most proteins in nature are chemically modified after they are made to control how, when, and where they function. The 3 core features of proteins are posttranslationally modified: amino acid side chains can be modified, peptide bonds can be cleaved or isomerized, and disulfide bonds can be cleaved. Cleavage of peptide bonds is a major mechanism of protein control in the circulation, as exempli...

The endoplasmic reticulum (ER) provides an environment optimized for oxidative protein folding through the action of Ero1p, which generates disulfide bonds, and Pdi1p, which receives disulfide bonds from Ero1p and transfers them to substrate proteins. Feedback regulation of Ero1p through reduction and oxidation of regulatory bonds within Ero1p is essential for maintaining the proper redox balan...

Protein disulfide bond is formed during post-translational modifications, and has been implicated in various physiological and pathological processes. Proper localization of disulfide bonds also facilitates the prediction of protein three-dimensional (3D) structure. However, it is both time-consuming and labor-intensive using conventional experimental approaches to determine disulfide bonds, es...

Animal toxins are the major class of secreted disulfide-rich proteins, with approximately 70% containing two or more disulfide bonds. Incorrect pairing of these disulfide bonds typically leads to a non-native three-dimensional fold accompanied by a loss of protein function. Determination of the native disulfide-bond framework is therefore a key component in the structural characterization of to...

Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cycl...

Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therap...