Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.

Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a K m ‫؍‬ 10 M. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.Proteins start life as linear amino acid chains and rapidly fold into compact active structures. Rapid folding is a prerequisite for the survival of a protein in the cell. It is not surprising then that critical steps in the folding process are assisted in the cell. One of the rate-limiting steps in protein folding is the formation of native disulfide bonds. In the cell, they form much more rapidly than in the test tube, implying that a catalyst is present in vivo (1-3).We and others have shown that there is a 21-kDa enzyme called DsbA that is essential for disulfide bond formation in vivo (4, 5). DsbA is a protein-folding catalyst that acts to form disulfides in newly synthesized periplasmic proteins. DsbA acts as the direct donor of disulfides to secreted proteins. The active site of DsbA consists of a pair of cysteines present in a CXXC motif that can oxidize to form a very reactive disulfide (6). This disulfide is rapidly transferred to proteins that are in the process of folding (7,8). The ease at which DsbA can be purified and manipulated biochemically and the availability of a 1.7-Å resolution crystal structure for DsbA have led to an abundance of information concerning the mechanism of DsbA action (9 -16). One of the features that is thought to contribute to the extreme oxidizing power of DsbA is the very low pK a of its Cys-30 residue, which makes it a superb leaving group in disulfide exchange reactions (13-16).In order for DsbA to act as a catalyst of disulfide bond formation, it needs to be reoxidized. Genetic studies strongly implicate an inner membrane protein called DsbB in the reoxidation of DsbA (17,18). Evidence that DsbB is required for the reoxidation of DsbA includes the observation that DsbA accumulates in a reduced form in DsbB mutants (18). The isolation from cells of a DsbA-DsbB dimer covalently linked by a disulfide bond implies a direct interaction between t...