Disulfide shuffling

Figure 3 Representative geometries of polypeptide chains where introduction of a cysteine poses high risk of intramolecular disulfide shuffling. (A) Within 1—3 residues from a native cysteine in a loop without defined secondary structure. (B and Q Within...

There are many approaches to producing functional recombinant chemokines from E. coli in addition to the ones referenced or presented here. The goal of this work is to describe a robust protocol we employ for many different chemokines, which draws from a variety of previously described approaches. We illustrate the impact of disulfide shuffling cocktails on the... 

The second mode of toxicity is postulated to involve the direct interaction of the epidithiodiketopiperazine motif with target proteins, forming mixed disulfides with cysteine residues in various proteins. Gliotoxin, for example, has been demonstrated to form a 1 1 covalent complex with alcohol dehydrogenase [13b, 17]. Epidithiodi-ketopiperazines can also catalyze the formation of disulfide bonds between proxi-mally located cysteine residues in proteins such as in creatine kinase [18]. Recently, epidithiodiketopiperazines have also been implicated in a zinc ejection mechanism, whereby the epidisulfide can shuffle disulfide bonds in the CHI domain of proteins, coordinate to the zinc atoms that are essential to the tertiary structure of that domain, and remove the metal cation [12d, 19],... 

Shuffle test. An enzyme that catalyzes disulfide-sulfhydryl exchange reactions, called protein disulfide isomerase (PDl), has been isolated. PDI rapidly converts inactive scrambled ribonuclease into enzymatically active ribonuclease. In contrast, insulin is rapidly inactivated by PDI. What does this important observation imply about the relation between the amino acid sequence of insulin and its three-dimensional structure ... 

Cysteine residues form disulfide bonds, which are important to protein structural integrity. Shuffling of these bonds, where two sulfur atoms from two different amino acid molecules link up, often changes the 3D structure, causing a loss of activity. 

Intermolecular disulfide bond formation between cysteinyl residues takes place at alkaline pH tmder oxidizing conditions. Proteins with reactive free thiol groups should be purified tmder reducing conditions (typically 1-10 mM reducing agent) in the presence of EDTA. Even proteins with disulfide bonds may participate in intermolecular disulfide bond reactions due to disulfide bond shuffling at neutral and alkaline pH. 

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