Protein engineering remains an area of growing importance in pharmaceutical and biotechnology research. Stabilization of a folded protein conformation is a frequent goal in projects that deal with affinity optimization, enzyme design, protein construct design, and reducing the size of functional proteins. Indeed, it can be desirable to assess and improve protein stability in order to avoid liab...

Disulfide bonds are ubiquitous in proteins. According to a recent survey, there are 97 741 disulfide bonds in 121 779 protein structures available in the Protein Data Bank (PDB).1 Native2,3 as well as engineered4-6 disulfide bonds have been shown to control the stability and function of proteins. The redox state of protein disulfide bonds in vivo, governing protein stability and function, depen...

Cellular compartments differ dramatically in their redox potentials. This translates directly into variations in the extent of disulfide bond formation within proteins, depending on their cellular localization. It has long been assumed that proteins that are present in the reducing environment of the cytosol do not possess disulfide bonds. The recent discovery of a number of cytosolic proteins ...

The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur...

Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond for...

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to exa...

It has been previously demonstrated that the vacuolar H+-ATPase (V-ATPase) of clathrin-coated vesicles is reversibly inhibited by disulfide bond formation between conserved cysteine residues at the catalytic site on the A subunit (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13224-13230). Proton transport and ATPase activity of the purified, reconstituted V-ATPase are now shown to be inh...

Elemental sulfur (S8) was found to react with [(TMPA)CuI(CH3CN)]+ to form the trans-mu-1,2 end-on disulfide complex [(TMPA)Cu-S-S-Cu(TMPA)]2+. The X-ray structure of this centrosymmetric disulfide complex shows a Cu(1)-S(1) bond length of 2.280(2) A and a S(1)-S(1A) bond length of 2.044(4) A.

P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) label...