**1. Introduction: covalent bonds in proteins**

The amino acids comprising a protein are covalently linked by peptide bonds, which collectively form the "backbone" of the protein. These bonds are the most prevalent covalent links

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between amino acids in proteins and form the foundations that support protein secondary and tertiary structures. In addition to peptide bonds, disulfide bonds are the second most common covalent bonds between amino acids. Disulfide bonds are sulfur-sulfur bonds formed within a protein when the thiol (-SH) groups of two cysteine residues are each oxidized, resulting in the net loss of two electrons to an oxidizing agent. Other names for these bonds include S-S bonds, disulfide bridges, disulfide crosslinks, and simply disulfides/disulphides. Disulfide bonds play three main roles in proteins as signal relays (signaling disulfide bonds), as active site residues in enzymatic catalysis (catalytic disulfide bonds), and as structural supports (structural disulfide bonds). Signaling disulfide bonds act as environmental redox readouts, such as those observed in the OxyR transcription factor (reactive oxygen species sensing), the ArcAB system (senses changes in respiratory growth conditions), and the antisigma factor RsrA (activates cytoplasmic oxidative stress response) [1–3]. Catalytic disulfide bonds act as active site residues in oxidoreductases such as DsbA and DsbC, which catalyze the oxidation and isomerization of disulfide bonds, respectively [4–6]. While signaling and catalytic disulfide bonds are important, the primary functions of structural disulfide bonds are to facilitate protein folding and to stabilize protein tertiary structure, which will be the focus of this chapter.

proteins is contained within its amino acid sequence [12–17]. Evidence supporting this idea now known as Anfinsen's dogma—was obtained from *in vitro* experiments in which RNase A was denatured and completely reduced to yield an unstructured polypeptide chain containing eight cysteine residues. Removal of denaturant and reducing agent and subsequent incubation of the enzyme open to atmosphere resulted in relatively rapid and spontaneous formation of disulfide bonds—formed by oxidation via molecular oxygen—followed by much slower recovery of RNase activity. Under optimized conditions, the halftime required for recovery of RNase activity was 20 min. This apparent lag phase between disulfide-bond formation and recovery of activity suggested that spontaneous disulfide-bond formation resulted in incorrect cysteine pairing and that the recovery of activity required additional time for disulfide interchange and proper formation to occur [16]. This also suggested that a system for oxidizing and reshuffling disulfide bonds existed *in vivo*, since the 20-min recovery of activity observed *in vitro* was far

From Biology to Biotechnology: Disulfide Bond Formation in *Escherichia coli*

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Shortly after demonstrating the spontaneous formation of disulfide bonds in reduced RNase A, Anfinsen and colleagues identified an enzyme, isolated from rat liver microsomes, that stimulated RNase A reactivation, with recovery of activity requiring a halftime of 4.5 min. This discovery confirmed the existence of an *in vivo* system capable of catalyzing the formation of the correct disulfide bonds to yield the active enzyme [18]. In addition to the rat liver, the same enzyme had been identified by Venetianer and Straub in chicken, pig, and pigeon pancreatic tissue [17]. Further studies in which the microsomal enzyme was incubated with reduced egg white lysozyme as a substrate—which contains four disulfide bonds required for its activity—demonstrated similar recovery of activity. Due to the recovery of activity in RNase A from both bovine pancreas and rat liver and in egg white lysozyme, the substrate specificity of the enzyme appeared to be rather low. As a result, this enzyme was speculated to be a general mediator of "sulfhydryl-disulfide exchange" *in vivo*. The catalyzed reaction is driven by the release of free energy attained upon folding substrates from unorganized conformations to their more stable native structures [18–20]. Furthermore, the enzyme catalyzing sulfhydryl-disulfide exchange enzyme was shown to possess a DTT-sensitive disulfide bond that was likely to be involved in catalysis [19]. In 1973, nearly 6 years after its identification, the microsome-associated enzyme that catalyzed sulfhydryl-disulfide exchange was given a

In the early 1980s, several studies demonstrated a correlation between PDI activity and the synthesis of disulfide-bond containing proteins in specialized cells and tissues [21, 22]. However, it was not until 1983—nearly 10 years after its naming—that PDI was purified to homogeneity and biochemically characterized [23]. Homogenous PDI was shown to catalyze the reduction of disulfide bonds in insulin *in vitro*. This result demonstrated that PDI could catalyze both disulfide-bond formation and reduction, and led some to question whether the name 'protein disulfide isomerase' was a misnomer. Based on the physiological evidence surrounding PDI—its distribution in tissue with great abundance of disulfide-bonded proteins [22]; its localization in the endoplasmic reticulum, where many disulfide-bonded proteins are synthesized; and its broad substrate specificity—in conjunction with findings showing PDI to

be a relatively poor reductase, the name PDI was retained and is still in use [24].

too slow to be operative in the cell.

name: protein disulfide isomerase (PDI).
