£\-Bungarotoxin (£\-Bgt), a £\-neurotoxin from Taiwan banded krait (Bungarus multicinctus), consists of a single polypeptide chain of 74 amino acid residues cross-linking with five disulfide bonds. In order to explore the structural dependence of the disulfide bonds in the toxin molecule, the mutants with deleting one out of the five disulfide bonds were prepared by site-directed mutagenesis. The mutated and wild-type cDNAs were subcloned into the expression vectors pET14b as well as pET32a(+), and then transformed into Escherichia coli, BL-21(DE3). The recombinant proteins derived from pET14b expression system were isolated from inclusion bodies of E. coli, and refolding into their folded structure in vitro. Alternatively, the proteins derived from pET32a(+) system were expressed as a fusion protein and purified on a His-Bind resin column. Moreover, the recombinant proteins were further purified using a reverse phase column, and the homogeneity of recombinant proteins was determined by SDS-PAGE analyses. Noticeably, the refolding reaction was beneficially achieved by deleting the disulfide bond Cys29-Cys33. Although SDS-PAGE analyses showed that the recombinant proteins were homogeneous in molecular weight, several disulfide isomers appeared in each protein preparation as revealed by native gel analyses. These observations suggest that the formation of disulfide bonds and folding of£\-Bgt may pass through multiple pathways.

Yes / The disulfide bond plays an important role in the formation and stabilisation of higher order structures of peptides and proteins, while in recent years interest in this functional group has been extended to carbohydrate chemistry. Rarely found in nature, glycosyl disulfides have attracted significant attention as glycomimetics, with wide biological applications including lectin binding, as key components of dynamic libraries to study carbohydrate structures, the study of metabolic and enzymatic studies, and even as potential drug molecules. This interest has been accompanied and fuelled by the continuous development of new methods to construct the disulfide bond at the anomeric centre. Glycosyl disulfides have also been exploited as versatile intermediates in carbohydrate synthesis, particularly as glycosyl donors. This review focuses on the importance of the disulfide bond in glycobiology and in chemistry, evaluating the different methods available to synthesise glycosyl disulfides. Furthermore, we review the role of glycosyl disulfides as intermediates and/or glycosyl donors for the synthesis of neoglycoproteins and oligosaccharides, before finally considering examples of how this important class of carbohydrates have made an impact in biological and therapeutic contexts. / The authors thank the Institute of Cancer Therapeutics (University of Bradford) Doctoral Training Centre for financial support.

Adiponectin, a hormone that homo-oligomerizes into trimer, hexamer, or higher molecular weight (HMW) species, is involved in maintaining insulin sensitivity in muscle and liver. Interestingly, its functions appear to be oligomer-specific. Recent data suggest that HMW levels are decreased in obesity and insulin resistance, although, the cause for this decrease is not known. Impaired assembly to the octadecamer represents one possible reason for decreased HMW adiponectin in insulin resistance and type 2 diabetes, but mechanisms by which HMW adiponectin assembles are unknown. This dissertation discusses the progress that we have made regarding formation of HMW adiponectin in vitro.I found that disulfide bonds are important in the assembly process to octadecameric adiponectin, but are not required for stability of the octadecamer itself. We showed that hydrogen peroxide accelerated oligomerization to the octadecamer through formation of disulfide bonds, while alkylation of the cysteines led to inhibition of both oligomerization and disulfide bond formation. Using comparative native/denaturing polyacrylamide gel electrophoresis (PAGE), dynamic light scattering, and tandem mass spectrometry, we demonstrated that octadecamer is stable in the absence of disulfide bonds by using multiple biochemical and biophysical assays. In addition, oxidized adiponectin oligomerizes to octadecamer far slower than reduced adiponectin. To further evaluate the role of disulfide bonds in the formation to octadecamer, we analyzed the role of reduction potential on adiponectin oligomerization. We observed that under immediate oxidizing conditions, hexamers and trimers form. Oxidized hexamer can form HMW adiponectin through disulfide bond rearrangement using beta-mercaptoethanol (βME) or increasing the total concentration of glutathione under oxidizing conditions. To further understand the role of disulfide bonds, we showed that zinc increased the oligomerization to octadecamer. This effect was associated with decreased initial disulfide bonding during the assembly to the octadecamer. In summary, these data suggest the rate of disulfide bond formation and the ability to undergo disulfide bond isomerization are important in the oligomerization process of HMW adiponectin.

Of the 20 standard amino acids, cysteines are the only amino acids that have a reactive sulphur atom, thus enabling two cysteines to form strong covalent bonds known as disulfide bonds. Even though almost all proteins have cysteines, not all of them have disulfide bonds. Disulfide bonds provide structural stability to proteins and hence are an important constraint in determining the structure of a protein. As a result, disulfide bonds are used to study various protein properties, one of them being protein folding. Protein structure prediction is the problem of predicting the three-dimensional structure of a protein from its one-dimensional amino acid sequence. Ab initio methods are a group of methods that attempt to solve this problem from first principles, using only basic physico-chemical properties of proteins. These methods use structure libraries of short amino acid fragments in the process of predicting the structure of a protein. The protein structures from which these structure libraries are created are not classified in any other way apart from being non-redundant. In this thesis, we investigate the structural dissimilarities of short amino acid fragments when occurring in proteins with disulfide bonds and when occurring in those proteins without disulfide bonds. We are interested in this because, as mentioned earlier, the protein structures from which the structure libraries of ab initio methods are created, are not classified in any form. This means that any significant structural difference in amino acids and short fragments when occurring in proteins with and without disulfide bonds would remain unnoticed as these structure libraries have both fragments from proteins with disulfide bonds and without disulfide bonds together. Our investigation of structural dissimilarities of amino acids and short fragments is done in four phases. In phase one, by statistically analysing the phi and psi backbone dihedral angle distributions we show that these fragments have significantly different structures in terms of dihedral angles when occurring in proteins with and without disulfide bonds. In phase two, using directional statistics we investigate how structurally different are the 20 different amino acids and the short fragments when occurring in proteins with and without disulfide bonds. In phase three of our work, we investigate the differences in secondary structure preference of the 20 amino acids in proteins with and without disulfide bonds. In phase four, we further investigate and show that there are significant differences within the same secondary structure region of amino acids when they occur in proteins with and without disulfide bonds. Finally, we present the design and implementation details of a dihedral angle and secondary structure database of short amino acid fragments (DASSD) that is publicly available. Thus, in this thesis we show previously unknown significant structure differences in terms of backbone dihedral angles and secondary structures in amino acids and short fragments when they occur in proteins with and without disulfide bonds.

Protamine, a set of small basic proteins (P1 and P2), play a key role in compacting and protecting the DNA in sperm. As such, the structure of how P1 and P2 bind to DNA and potentially themselves and each other is of interest to several fields including forensics. In forensic DNA analysis, protamine binding of DNA is taken advantage of in the “differential extraction” procedure in which a sample that contains sperm and non-sperm cells can have DNA from the two different cell types separated and extracted at different points thus preventing a mixture of DNA. A key component of this greater structure and what makes the differential extraction functional are the disulfide bonds formed by protamine. So as a first step to elucidating the protamine-DNA complex, methods to analyze human protamine via 2D-Liquid Chromatography-Mass Spectrometry (2D-LC-MS) were developed in the hopes they could be used for disulfide bond mapping. Methods and multiple strategies for digestion, 2D-LC-MS were investigated and developed using chum salmon protamine. Digestion strategies were developed for Chymotrypsin and Lys-C, Trypsin or Arg-C with incubation times and substrate:enzyme mass ratios optimized. Various “trap and elute” 2D-chromatography configuration were tested for analysis intact and digested protein. Using H2O with 2% NH4OH as the loading mobile phase and H2O and Acetonitrile both with 0.5% formic acid as the eluting mobile phases with the first dimension column being an HLB (Hydrophilic Lipophilic Balance) 2.1 x 30 mm column and the second dimension being an C18 2.1 x 100 mm was found to produce the highest signal.

A global analysis of the human proteome demonstrates that there are ~5500 tryptic fragments that contain four cysteines in close proximity. Elucidating whether they form disulfide bonds in vivo under different conditions is particularly important because cysteines are known to be a vital cellular redox sensor as well as a catalytic site for important biochemical reactions. However, currently there are no methods that can resolve disulfide patterns in closely-packed cysteine residues from a complex sample. In order to address this problem, we have developed a novel mass-spectrometry-based method to identify the different disulfide bonding patterns possible, using SNAP25B cysteine-rich region as a test case. Unlike traditional proteomics, this method uses non-reduced sample preparation, thus preserving intact disulfide bonds. It relies on collision-induced dissociation (CID) to cause double-backbone and heterolytic disulfide-bond cleavage and compares this to the theoretical MS/MS spectra. CID in an ion trap gives robust detection of double backbone cleavages and heterolytic disulfide-bond cleavages. Here, we report, for the first time, identification of all three disulfide patterns for double-disulfide species of SNAP25B using collision-induced dissociation.

Enterococci are found in soil, sewage, food, water, and are commensal to the gastrointestinal tracts of mammals, insects, and birds. Enterococci often become nosocomial pathogens that cause a wide variety of diseases including urinary tract infections, endocarditis, and septicemia. These infections are often difficult to treat with antibiotics because most of the nosocomial strains are multi-drug resistant. Enterococcal plasmids function as reservoirs for resistance genes because they are extremely stable, allow for specific and efficient transfer, and can acquire resistance determinants from the chromosome and other plasmids. Additionally, enterococcal plasmids transfer across species boundaries transferring resistance genes like vancomycin to species like Staphylococcus aureus. There are two types of enterococcal plasmids, pheromone-responsive and broad host range. Pheromone-responsive plasmids are extremely stable, have a limited host range, and are primarily found in Enterococcus faecalis. Broad host range plasmids of E. faecalis and Enterococcus faecium are less stable than pheromone-responsive plasmids, but have an expanded host range into other Gram-positive species. E. faecalis has at least 25 known pheromone-responsive conjugative plasmids. One of the most extensively studied pheromone-responsive conjugative plasmids, pCF10. Conjugation of pCF10 from donor to recipient cell is induced by pheromone cCF10. cCF10 is contained within n the lipoprotein signal sequence encoded by the E. faecalis chromosomal gene ccfA. The lipoprotein signal sequence is processed by a series of proteolytic cleavage events to produce mature cCF10. Maturation of pheromone cCF10 produces three peptides: pre-cCF10 (CcfA1-22), cCF10 (CcfA13-19), and CcfA1-12. Cells containing pCF10 continue to produce cell membrane associated precursor pheromone of cCF10 (pre-cCF10), as well as, secreted and cell wall-associated cCF10. The presence of cCF10 does not self-induce conjugation by the donor cell because of two inhibitory molecules, PrgY and iCF10. Transmembrane protein PrgY is encoded by pCF10 and reduces cell wall associated cCF10, iCF10 is a pCF10 encoded inhibitory peptide (AITLIFI) that binds to PrgX, preventing cCF10 binding. While cCF10 controls pCF10 conjugation, pre-cCF10 controls host range of pCF10 by interacting with pCF10 replication initiation protein PrgW. cCF10 can initiate conjugation and mobilize the transfer of plasmids into other species, including Lactococcus lactis, but pCF10 cannot be maintained within the cell. However, if L. lactis is engineered to produce pre-cCF10, pCF10 can be maintained. The pre-cCF10 involvement in the establishment of pCF10 into other species might be related to the observation that it binds to the pCF10 replication initiation protein PrgW. By in vitro affinity chromatography experiments, interaction of cCF10 and pre-cCF10 with PrgW induced changes in PrgW mobility in gel electrophoresis that caused by formation of doublets and formation of aggregates which were thought to be mediated by disulfide bonds. Initial evidence of regulation of PrgW conformation by disulfide bonds was seen in Western blots of E. faecalis whole cell lysates where PrgW migration is sensitive to reduction. Sequence alignment comparisons between PrgW and a group of 54 of 59 known RepA_N superfamily proteins in E. faecalis revealed three highly conserved cysteines; these RepA_N proteins had a limited host range to E. faecalis. To study the importance of theses cysteines in pCF10 maintenance and host range limitation, prgW single, double, and triple cysteine to alanine (C to A) substitutions were generated. The cysteine mutant prgW was cloned into a plasmid functioning as either a contained the prgW alone (pORI10), or containing prgW with genes necessary for efficient pCF10 maintenance (pMSP6050). While all cysteine mutant plasmids of pORI10 and pMSP6050 were still capable of replicating in E. faecalis, the plasmid stability and copy number decreased, providing evidence that the cysteines were important to PrgW function. Additionally, Western blot analysis revealed PrgW C to A substitutions decreased PrgW aggregation. Mutations of PrgW cysteines reduced pMSP6050 stability and aggregation, but increased host range to L. lactis. Both L. lactis engineered to produce pre-cCF10 and the mutation of the conserved cysteines of PrgW extended host range of pMSP6050 into L. lactis. These data taken together with the observations that pre-cCF10 induced PrgW aggregation suggested that pre-cCF10 regulated the activity of the PrgW replication initiation protein through disulfide bonds. While the conserved cysteines of RepA_N proteins are found only in E. faecalis, phylogenetic analysis revealed that RepA_N homologs lacking the three cysteines are also found in E. faecium or S. aureus, suggesting that the host range of multiple plasmids might be affected by cysteine bond formation. Phylogenetic analysis also showed that the RepA_N proteins of enterococci and staphylococci appear to have evolved to determine host range based on the presence of two of the three conserved cysteines. Modular evolution of E. faecalis plasmids, like pCF10, that contained RepA_N proteins with three conserved cysteines, might have determined the fate of the plasmid as a limited host range, stable reservoir for antibiotic resistance. / Microbiology and Immunology

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

Disulfide bonds play important roles in establishing and stabilizing three-dimensional protein structure, and mass spectrometry (MS) has become the primary detection method to decipher their biological and pathological roles. Several experimental methods before or after MS detection have been developed to aid in disulfide bond assignment, such as tandem MS followed by database searching or modification of the disulfide bond via chemical reduction or oxidation. Despite these technological advancements, the detection and proper assignment of disulfide bonds have remained experimentally difficult. Therefore, we have developed an alternative method for disulfide bond elucidation using capillary electrophoresis-mass spectrometry (CE-MS) combined with an on-target performic acid oxidation method for matrix assisted laser desorption/ionization (MALDI) deposited samples. An information rich CE-MS method that results in distinct charge-state trends observed in two-dimensional plots of log(mu eff) versus log (MW) was developed to enhance the confidence of peptide and protein identifications. The charge-state trends provide information about the number of basic amino acid residues present within each peptide. This information can be used to develop methods to screen for posttranslationally modified peptides (e.g., phosphorylation, disulfide bonds, etc.). In the case of disulfide bonds, the highly charged peptides (i.e., 3, 4 or greater charge states) have a high probability of being disulfide-linked peptides, owing to charge contribution of both peptides forming the disulfide bridged peptide. However, intra-linked disulfide bridged peptides can also be present at lower charge states. Therefore, a chemically selective method to rapidly locate disulfide-linked peptides that have been separated by CE-MS must be developed. An on-target performic acid oxidation method was developed to provide the chemical selectivity towards disulfide bonds, i.e., converting the cystine bond to form two peptides modified with a cysteic acid (SO3H) side chain. The on-target oxidation method offers (i) no post-oxidation sample cleanup, (ii) improved throughput over solution-phase oxidation methods, and (iii) easily adapted to CE separations coupled offline with MALDI-MS. The evaluation of the on-target oxidation experimental parameters, the fragmentation behavior of cysteic acid-containing peptides and an alternative method for disulfide bond elucidation, using CE-MS combined with the ontarget oxidation method, are discussed within.

Disulfide bond formation is an essential process for the folding and biological activity of most extracellular proteins; however, it may become the limiting step when the production of these proteins is attempted in heterologous hosts such as Escherichia coli. The rearrangement of incorrect disulfide bonds between cysteines that do not normally interact in the native structure of a protein is carried out by disulfide isomerase enzymes. The disulfide isomerase present in the bacterial secretory compartment (the periplasmic space) is the homodimeric enzyme DsbC. The objective of this dissertation was to understand the key features of how DsbC catalyzes disulfide bond isomerization. Chimeric disulfide isomerases comprising of protein domains that share a similar function, or are homologous to domains of DsbC were constructed in an effort to understand the effect of the domain orientation in the dimeric protein, and the need for a substrate binding region in disulfide isomerases. We successfully created a series of fusion enzymes, FkpA-DsbAs, which catalyze in vivo disulfide isomerization with comparable efficiency to DsbC. These enzymes comprise of the peptide binding region of the periplasmic chaperone FkpA, which is functionally and structurally similar to the binding domain of DsbC but share no amino acid homology with it, fused to the bacterial oxidase DsbA. In addition, these chimeric enzymes were shown to assist in the initial formation of disulfide bonds, a function that is normally exhibited only by DsbA. Directed evolution of the FkpA-DsbA proteins conferred improved resistance to CuCl₂, a phenotype dependent on disulfide bond isomerization and highlighted the importance of an optimal catalytic site. The bacterial disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers and two opposing catalytic domains. The functional significance of the existence of two catalytic domains of DsbC is not well understood yet. The fact that identical subunits naturally dimerize to generate DsbC has so far limited the study of the individual catalytic sites in the homodimer. In chapter 3 we discuss the engineering, in vivo function, and biochemical characterization chapter 3 we discuss the engineering, in vivo function, and biochemical characterization of DsbC variants covalently linked via (Gly3Ser) flexible linkers. We have either inactivated one of the catalytic sites (CGYC), or entirely removed one of the catalytic domains while maintaining the putative binding area intact. Our results support the hypotheses that dual catalytic domains in DsbC are not necessary for disulfide bond isomerization, but are important in terms of increasing the effective concentration of catalytic equivalents, and that the availability of a substrate binding region is a determining feature in isomerization. Finally, we have carried out initial studies to map the residues and sequence motifs that are recognized in substrate proteins that interact with DsbC. Although the main putative binding region of DsbC has been localized within the limits of the hydrophobic cleft that emerges from the interaction of the N-terminal domains of this enzyme, and, a few native substrates have already been identified, no information on the features of substrate proteins that are recognized by the enzyme has been reported. To address this problem, we have screened two different, 15 amino-acid random peptide libraries for binding to DsbC. We have successfully isolated several peptides with high affinity for the enzyme. Possible consensus binding motifs were identified and their significance in substrate recognition will be examined in future studies. / text