Martin W. Bader

Oxidative Protein Folding Is Driven by the Electron Transport System

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.

Turning a disulfide isomerase into an oxidase: DsbC mutants that imitate DsbA

There are two distinct pathways for disulfide formation in prokaryotes. The DsbA‐DsbB pathway introduces disulfide bonds de novo, while the DsbC‐DsbD pathway functions to isomerize disulfides. One of the key questions in disulfide biology is how the isomerase pathway is kept separate from the oxidase pathway in vivo. Cross‐talk between these two systems would be mutually destructive To force communication between these two systems we have selected dsbC mutants that complement a dsbA null mutation. In these mutants, DsbC is present as a monomer as compared with dimeric wild‐type DsbC. Based on these findings we rationally designed DsbC mutants in the dimerization domain. All of these mutants are able to rescue the dsbA null phenotype. Rescue depends on the presence of DsbB, the native re‐oxidant of DsbA, both in vivo and in vitro. Our results suggest that dimerization acts to protect DsbCs active sites from DsbB‐mediated oxidation. These results explain how oxidative and reductive pathways can co‐exist in the periplasm of Escherichia coli.

RECONSTITUTION OF A PROTEIN DISULFIDE CATALYTIC SYSTEM

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 dsbBnull 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 μm. 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.

DsbC activation by the N-terminal domain of DsbD

The correct formation of disulfide bonds in the periplasm of Escherichia coli involves Dsb proteins, including two related periplasmic disulfide-bond isomerases, DsbC and DsbG. DsbD is a membrane protein required to maintain the functional oxidation state of DsbC and DsbG. In this work, purified proteins were used to investigate the interaction between DsbD and DsbC. A 131-residue N-terminal fragment of DsbD (DsbDα) was expressed and purified and shown to form a functional folded domain. Gel filtration results indicate that DsbDα is monomeric. DsbDα was shown to interact directly with and to reduce the DsbC dimer, thus increasing the isomerase activity of DsbC. The DsbC–DsbDα complex was characterized, and formation of the complex was shown to require the N-terminal dimerization domain of DsbC. These results demonstrate that DsbD interacts directly with full-length DsbC and imply that no other periplasmic components are required to maintain DsbC in the functional reduced state.

Catalysis of disulfide bond formation and isomerization in Escherichia coli

Publisher Summary This chapter deals with folding catalysts, in particular with catalysts that are essential for disulfide bond formation in Escherichia coli. Nevertheless, some of these catalysts contain chaperone activity, demonstrating that these two activities are sometimes found within the same protein molecule. The folding of proteins into their three-dimensional structure is essential for their biological function. For proteins that contain disulfide bonds, formation of these bonds is often an important step in the folding reaction. DsbA is identified by the use of a disulfide indicator protein, MalF-fi-galactosidase. This fusion protein lacks fl-galactosidase activity when present in a wild-type E. coli background that is competent in forming disulfides. Despite their common structures, thioredoxin and DsbA fulfill different functions and exist in different cellular compartments. While thioredoxin acts as a reductant of disulfide bonds in the cytosol introduces disulfide bonds into newly synthesized proteins during their translocation to the periplasm. The small equilibrium constant of DsbA with glutathione demonstrates that the disulfide bond formed by DsbA is highly unstable. The stability of a particular disulfide bond corresponds to the extent to which a protein is stabilized by this bond. In other words, the more stable the disulfide bond, the more stable the protein conformation.

Protein oxidation: prime suspect found 'not guilty'.

Glutathione has long been suspected to be the primary source of oxidative power for protein folding. It has now been shown to be just the opposite, namely a source of reductants. The ultimate origin of oxidants has become even more of a mystery.

Protein oxidation: prime suspect found |[lsquo]|not guilty|[rsquo]|

Glutathione has long been suspected to be the primary source of oxidative power for protein folding. It has now been shown to be just the opposite, namely a source of reductants. The ultimate origin of oxidants has become even more of a mystery.