α-Conotoxins are peptide neurotoxins isolated from the venom ducts of carnivorous marine cone snails that exhibit exquisite pharmacological potency and selectivity for various nicotinic acetylcholine receptor subtypes. As such, they are important research tools and drug leads for treating various diseases of the central nervous system, including pain and tobacco addiction. Despite their therape...

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this "mechanopresentation" rema...

The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV...

We used recombinant DNA technology to construct a mutant form of Pseudomonas exotoxin A (PE) called cysPE35 that contains amino acids 280-364 and 381-613 of PE. cysPE35 begins at the native PE proteolytic cleavage site and contains a single cysteine residue at position 287 that can be used to conjugate the toxin to monocional antibodies (MAbs). Unlike immunotoxins containing larger mutant forms...

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalyti...

Furthermore, the assignment of disulfide linkages sometime is inconclusive when a protein contains multiple cysteines due to the significantly increased number of possible disulfide-bonded peptide isomers. For example, there are totally 105 possible disulfide-bond pairing schemes for a protein containing eight cysteine residues (while an IgG1 mAb typically has 32 or more cysteines in its two li...

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for ...

Escherichia coli thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Aslund, and J. Beckwith, EMBO J. 17:5543-5550, 1998). We recently showed that when a signal sequence...

UNLABELLED We investigated whether there is any association between a native-like conformation and the presence of only the canonical (i.e., native) disulfide bonds in the gp120 subunits of a soluble recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein. We used a mass spectrometry (MS)-based method to map the disulfide bonds present in nonnative uncleaved gp140 pr...

Heparanase is overexpressed in many solid tumor cells and is capable of specifically cleaving heparan sulfate, and this activity is associated with the metastatic potential of tumor cells; however, the activation mechanism of heparanase has remained unknown. In this study, we investigated the link between disulfide bond formation and the activation of heparanase in human tumor cells. Mass spect...