**2. Disulfide-bond research during the pre-genomic sequencing era**

The pre-genomic sequencing era focuses on the research surrounding disulfide-bond formation conducted in the early 1960s through the early 1980s. The work of Anfinsen, Straub, and others on the disulfide-bond formation in Ribonuclease A (RNase A) provided the initial impetus behind studying this process and is described below.

The roles of structural disulfide bonds in protein folding and stability are well exemplified and characterized in the protein RNase A, which contains a total of four disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys74) that contribute to the "legendary" stability of RNase A [7]. Indeed, the classic isolation and purification protocol of active RNase A from bovine pancreas calls for extremely harsh conditions in the context of proteins: treatment of pancreatic tissue with 0.25 *N* sulfuric acid at 5°C for 1 day, followed by ammonium sulfate precipitation and boiling in 20% saturated ammonium sulfate at pH 3.0 for 5–10 min [8]. While these purification conditions disrupt noncovalent interactions, the covalent nature of disulfide bonds allows them to survive such treatments, thereby imparting structural stability to the protein. Disulfide bonds also constrain the number of conformations an unfolded protein can adopt, which destabilizes the unfolded state relative to the folded state [9]. In fact, the stability of properly folded RNase A has been estimated to be 8 kcal/ mol greater than the unfolded state [10], and the four disulfide bonds collectively contribute an estimated 19 kcal/mol to this stability [11]. While it is clear that structural disulfide bonds play important roles in maintaining the overall integrity of a protein, especially in the case of RNase A, they also serve to aid in the initial folding and refolding of a protein.

Seminal experiments conducted in the early 1960s on RNase A led to the idea that all of the information required to form the correct structures and disulfide bonds of small, globular 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 too slow to be operative in the cell.

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

360 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

and to stabilize protein tertiary structure, which will be the focus of this chapter.

RNase A, they also serve to aid in the initial folding and refolding of a protein.

Seminal experiments conducted in the early 1960s on RNase A led to the idea that all of the information required to form the correct structures and disulfide bonds of small, globular

impetus behind studying this process and is described below.

**2. Disulfide-bond research during the pre-genomic sequencing era**

The pre-genomic sequencing era focuses on the research surrounding disulfide-bond formation conducted in the early 1960s through the early 1980s. The work of Anfinsen, Straub, and others on the disulfide-bond formation in Ribonuclease A (RNase A) provided the initial

The roles of structural disulfide bonds in protein folding and stability are well exemplified and characterized in the protein RNase A, which contains a total of four disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys74) that contribute to the "legendary" stability of RNase A [7]. Indeed, the classic isolation and purification protocol of active RNase A from bovine pancreas calls for extremely harsh conditions in the context of proteins: treatment of pancreatic tissue with 0.25 *N* sulfuric acid at 5°C for 1 day, followed by ammonium sulfate precipitation and boiling in 20% saturated ammonium sulfate at pH 3.0 for 5–10 min [8]. While these purification conditions disrupt noncovalent interactions, the covalent nature of disulfide bonds allows them to survive such treatments, thereby imparting structural stability to the protein. Disulfide bonds also constrain the number of conformations an unfolded protein can adopt, which destabilizes the unfolded state relative to the folded state [9]. In fact, the stability of properly folded RNase A has been estimated to be 8 kcal/ mol greater than the unfolded state [10], and the four disulfide bonds collectively contribute an estimated 19 kcal/mol to this stability [11]. While it is clear that structural disulfide bonds play important roles in maintaining the overall integrity of a protein, especially in the case of 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 name: protein disulfide isomerase (PDI).

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].

The advent of genome sequencing and PCR in the later 1980s caused a shift from eukaryotic PDI studies to research centered on bacterial disulfide-bond formation, which is detailed in the following section. It should be noted that Anfinsen's idea that the amino acid sequence of a protein encodes all of the information necessary for its proper folding was not fully correct. Even though Anfinsen shared the 1972 Nobel Prize in Chemistry with Stanford Moore and William H. Stein, the following decades of his and others' research showed that disulfide-bond formation and protein folding are, in fact, catalyzed processes *in vivo*. The work surrounding RNase A refolding and the elucidation of PDI serves as an example wherein the true answers to fundamental questions often require far more research to unravel their complexities.

(see **Figure 1**). Specifically, the DsbA protein transfers disulfide bonds to substrate proteins in the periplasm by accepting electrons from the substrate's cysteine residues. As a result, the cysteine residues of DsbA become reduced and the protein must be oxidized for it to catalyze another round of disulfide bond transfer [28, 29]. This oxidation reaction is carried out by DsbB, an inner membrane protein with two pairs of redox-active cysteines [30, 32]. The electrons received by DsbB in its oxidation of DsbA are transferred to the pool of quinones within the inner membrane [37, 39–43]. Then, the reduced quinones are recycled by cytochrome and terminal oxidases of the electron transport chain [42, 44–46]. Together, DsbA and DsbB act as the oxidation system for disulfide-bond formation in the periplasm (**Figure 1**). These two proteins form one part of the periplasmic disulfide-bond forming pathway; additional proteins, DsbC and DsbD, among others, play downstream roles in the fidelity of native

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

http://dx.doi.org/10.5772/67393

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**Figure 1.** The disulfide-bond-forming pathways in the periplasm of E. coli*.* A protein containing four cysteines in their reduced (free thiol) states is translocated into the periplasm by the SecYEG translocon. (1). Oxidized DsbA catalyzes disulfide-bond formation either as the protein is translocated or after, resulting in sequential disulfide bonds in this protein. DsbA is then oxidized to its active state by DsbB. DsbB is oxidized by ubiquinone or menaquinone under aerobic or anaerobic conditions, respectively (not shown). (2). If the disulfide bonds formed by DsbA are misoxidized, reduced DsbC catalyzes their isomerization to yield the properly folded protein. (3). DsbD then reduces DsbC to its active state. DsbD is reduced by an electron cascade originating from NADPH and mediated by thioredoxin reductase

disulfide bonds.

and thioredoxin in the cytoplasm (not shown).
