ABSTRACT
The increase in the number of projects carried out in protein crystallography laboratories has emphasized the need for effective management of project information and data. To meet this need, a flexible web-accessible database for protein crystallography project management (LISA) has been developed using the open-source software MySQL and PHP4. The database contains information about all aspects of structure-determination projects, including primer and plasmid sequences, protein expression, purification and crystallization results, structure coordinate files and resultant publications. The database web pages include links to relevant servers and contain, in addition, tools for processing stored information. The software package is freely available.
Subject(s)
Crystallography , Databases, Factual , Proteins/chemistry , Software , Documentation , Forecasting , Information Management , InternetABSTRACT
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 (DsbDalpha) was expressed and purified and shown to form a functional folded domain. Gel filtration results indicate that DsbDalpha is monomeric. DsbDalpha was shown to interact directly with and to reduce the DsbC dimer, thus increasing the isomerase activity of DsbC. The DsbC-DsbDalpha 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.
Subject(s)
Bacterial Proteins/metabolism , Escherichia coli/enzymology , Membrane Proteins/metabolism , Protein Disulfide-Isomerases/metabolism , Bacterial Proteins/chemistry , Cysteine/chemistry , Dimerization , Macromolecular Substances , Membrane Proteins/chemistry , Models, Molecular , Oxidation-Reduction , Protein Conformation , Protein Disulfide-Isomerases/chemistry , Protein Structure, TertiaryABSTRACT
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 DsbC's active sites from DsbB-mediated oxidation. These results explain how oxidative and reductive pathways can co-exist in the periplasm of Escherichia coli.
Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Oxidoreductases/metabolism , Protein Disulfide-Isomerases/metabolism , Bacterial Proteins/genetics , Membrane Proteins/genetics , Models, Molecular , Mutagenesis , Oxidation-Reduction , Oxidoreductases/genetics , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/genetics , Protein Structure, SecondaryABSTRACT
The protein disulfide bond isomerase DsbC catalyzes the rearrangement of incorrect disulfide bonds during oxidative protein folding in the periplasm of Escherichia coli. The active site cysteines of DsbC are maintained in the active reduced form by the transmembrane electron transporter DsbD. DsbD obtains electrons from the cytoplasm, transports them across the inner membrane, and passes them onto periplasmic substrates, such as DsbC. The electron transport process involves several thiol disulfide exchange reactions between different classes of thiol oxidoreductase. We were able to trap the final electron transport reaction using active site mutants yielding a stable DsbC-DsbDalpha complex. This disulfide cross-linked complex was purified to homogeneity and crystallized. Dehydration of the tetragonal crystals changed the unit cell dimensions from a approximately b = 73 A, c = 267.5 A to a = b = 68.9 A, c = 230.3 A, reducing the cell volume by 23% and the solvent content from 55 to 41%. Crystal dehydration and cryo-cooling improved the diffraction quality of the crystals from 7 to 2.3 A resolution.
Subject(s)
Protein Disulfide-Isomerases/chemistry , Crystallization , Crystallography, X-Ray , Protein Binding , Protein Conformation , Protein Disulfide-Isomerases/metabolismABSTRACT
DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.
