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Schumacher, F. F. "Functional bridging of protein disulfide bonds with maleimides." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1382399/.
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The application of chemical methods to biological systems has led to great advances in all life sciences and the discovery of novel approaches for therapy and diagnosis. Pivotal amongst these methods is the ability to chemically modify proteins to enhance their biophysical properties or add new functionality. Despite the success, the chemical toolbox of efficient and widely applicable protocols is relatively limited. In the work presented in this thesis the idea of protein modification via the targeting of solvent accessible disulfide bonds is explored. These are fairly common in secreted proteins and their reduction affords two cysteine residues, which are highly reactive nucleophiles. However, to preserve their native function – stabilisation and maintenance of the protein structure – ideally bis-reactive compounds are used that react simultaneously with both thiols and keep the covalent connection of the disulfide bond intact. To this end a selection of maleimides substituted with good leaving groups in the 3 and 4 positions as well as their N-functionalised versions were synthesised and tested for their reactivity. The findings were transferred to the small peptide hormone somatostatin, which served as a model system to explore kinetics and feasibility of the proposed “functionalisation by bridging”. Changing the leaving groups from halides to thiols enabled the development of in situ protocols where the bridging reagent could be employed in tandem with the reducing agent, greatly decreasing the reaction time and unwanted side reactions such as dimerisation or unfolding. The developed methods were then utilised to bridge the cystines of insulin as well as a selection of full length antibodies and antibody fragments. PEGylated, biotinylated, fluorescent or spin labelled analogues of these proteins were also synthesised. The biological activity, stability and functionality of the conjugates were assessed in biochemical and biophysical assays. Overall the functionalisation of disulfide bonds with maleimides was found to be site-specific, fast, high yielding and the inserted bridge was stable under various conditions. The modification was well tolerated and all prepared analogues exhibited the desired functionality. The scope and potential of present and future applications of the method are discussed.
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Eklund, Aron Charles 1974. "Patterns in the sequence context of protein disulfide bonds." Thesis, Massachusetts Institute of Technology, 2001. http://hdl.handle.net/1721.1/16804.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, February 2002.Includes bibliographical references (leaves 60-62).
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Disulfide bonds play an important role in the structural stability of the proteins that contain them. Yet, little is known about the specificity with which they are formed. To address this, a representative set of disulfide bonds from nonhomologous eukaryotic polypeptides was created. The amino acid sequences flanking these disulfide bonds were searched for conserved patterns that may reflect recognition sites by the disulfide bond forming enzyme protein disulfide isomerase (PDI). Several methods of classifying disulfide bonds were explored, and each class was analyzed for conserved sequence patterns. To maximize the chances of finding a conserved recognition site, a simulated annealing algorithm was implemented to divide a set of disulfide-bonded cysteines into two sets of cysteines with an average sequence environment that is as far from randomly-distributed as possible. No significant conserved patterns were found in the set of disulfide bonds or within any of the classification schemes introduced. Additionally, several methods for predicting disulfide bond connectivity were explored. The most successful methods predicted connectivity based on the sequential distance between cysteines.
by Aron Charles Eklund.
S.M.
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Baldus, Ilona Beatrice [Verfasser], and Peter [Akademischer Betreuer] Comba. "Mechanochemistry of Disulfide Bonds in Proteins / Ilona Beatrice Baldus ; Betreuer: Peter Comba." Heidelberg : Universitätsbibliothek Heidelberg, 2013. http://d-nb.info/1177040786/34.
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Bracchi, Michael Edward. "Exploring the orthogonal dynamic covalent imine and disulfide bonds in polymer systems." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3989.
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In the field of supramolecular systems chemistry the desire of researchers to endow functional macromolecular systems with orthogonal stimuli-responsiveness has fueled interest in the incorporation of multiple orthogonal dynamic covalent chemistries within a single chemical system. Dynamic covalent chemistry involves reversible bond forming processes which can be harnessed in the development of dynamic supramolecular systems providing a mechanism for structural reconfiguration of system components in response to external stimuli. Despite the inspiring gamut of recently brandished studies, there is a still a call on the resourcefulness of chemists to expand the capacity for the introduction of multi-faceted, orthogonal, stimuli-responsive behaviors. Herein, a critical analysis of recently reported landmark studies has been undertaken wherein the utility of orthogonal dynamic covalent bonding motifs in functional systems is highlighted. Furthering the concepts of orthogonality with respect to dynamic covalent chemistry presented therein, a small molecule ‘model system’ was developed with which the orthogonality of imine and disulfide dynamic covalent bonds was demonstrated by its operation. A key focus of this preliminary work was the orthogonal bond forming and bond breaking processes of imine and disulfide dynamic covalent bonds. Upon the establishment of conditions necessary to exploit the orthogonal utility of imine and disulfide bonds, the incorporation of aldehydes, amines and thiols within acrylamide-based copolymers as pendant functional groups was achieved. These pre-formed functional polymer building blocks were shown to undergo stimuli-responsive intermolecular cross-linking in aqueous media yielding disulfide or imine cross-linked nanoparticles or hydrogels. The scope and utility of imine and disulfide bonds in the formation of these nanostructured materials is compared and contrasted. It is reasoned that expanding understanding and availability of dynamic covalent bonding motifs will facilitate evolution of systems of greater sophistication capable of embodying increasingly information-rich processes.
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Merkel, Brian J. "Characterization of fibroblasts with a unique defect in processing antigens with disulfide bonds." VCU Scholars Compass, 1994. http://scholarscompass.vcu.edu/etd/5076.
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A Chinese hamster ovary (CHO) fibroblast, transfected with murine major histocompatibility complex (MHC) class II genes, inefficiently simulated CD4+ Th cells specific for ovalbumin (OVA), hen egg lysozyme (HEL), and pork insulin which contains disulfide bonds. However, the fibroblasts elicited a T cell response to λ-repressor, which lacks disulfide bonds, and efficiently presented synthetic peptides. A somatic cell hybrid WALC, generated by fusing the hamster fibroblast with a murine L cell fibroblast, very efficiently processed OVA and HEL, suggesting that impaired processing was genetically complemented, suggesting that the processing defect is a recessive trait. Three distinct processing phenotypes were observed among twenty-eight hybrid clones analyzed for their ability to process a suboptimal concentration of OVA suggesting that a limited number of genes mediates the defect of WAB4 cells. The hamster fibroblasts were capable of processing two distinct denatured forms of OVA and carboxymethylated HEL either as effectively or more efficiently than a B lymphoma cell. The CHO cells also displayed diminished disulfide reduction of an endocytosed conjugate consisting of 125I-tyramine linked to poly-(D-lysine) through a disulfide spacer compared with that of the cell hybrid, providing direct evidence for defective reductive cleavage for the CHO cells. Diminished aspartic acid-mediated proteolysis of Ag could not account for the phenotype, because cell lysates and separated organelles from the fibroblast possessed higher acidic aspartyl proteolytic activity than lysates and organelles from a B lymphoma cell. The WAB4 cells had normal intracellular levels of cysteine, however they possessed diminished levels of intracellular glutathione (GSH). Buthionine sulfoximine (BSO) - mediated reduction of intracellular levels of GSH decreased the ability of the hybrid line WALC to process HEL. Conversely, treatment of WAB4 cells with N-acetyl cysteine increased their efficiency in the processing of HEl. These findings indicate that the intracellular level of GSH influences the capacity of cells to process antigens with the disulfide bonds. Thus, the antigen processing defect exhibited by transfected CHO cells is probably caused by their impaired ability to reduce disulfide bonds which may be related to the diminished intracellular GSH level.
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Bewley, Kathryn Duffy. "Characterization of electron-transfer proteins: archaeal disulfide bonds and bacterial multi-heme cytochromes c." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12715.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Electron-transfer proteins that are responsible for redox homeostasis and long. range electron transfer are vital to intracellular and extracellular processes. In this thesis, several examples of electron-transfer proteins are studied in order to determine the emergent properties of multi-electron transfer chemistry. Thioredoxin (Trx) is a small redox-active protein that functions via its disulfide bond. These disulfides, characterized by a CXXC motif, are found to have a range of redox potentials that are often linked to function. Chapter 2 uses a set of archaeal thioredoxins from Thermoplasma acidophilum and Archaeoglobus fulgidus to test the current hypotheses using protein film voltammetry and solution-based experiments that examine folding energies. Thioredoxin reductase (TrxR) functions to provide reducing equivalents to Trx to keep it active in the cell. The TrxR from Thermoplasma acidophilum has been noted to be unusual in that it does not use NADPH as a reductant, as found in most TrxRs. The reaction between T. acidophilum Trx and TrxR is explored in Chapter 3 and a bioinfonnatic analysis of TaTrxR is included in Chapter 4 to better understand its relationship in the TrxR protein family, as well as attempt to identity its native reductant. In Chapter 5, the periplasmic decaheme cytochrome DmsE from Shewanella oneidensis is biochemically characterized. This protein is part of the dimethyl sulfoxide reduction pathway and is compared with MtrA, the well-studied decaheme protein from the dissimilatory metal reduction pathway in Shewanella. Additionally, a Cytoscape analysis of the MtrA/DmsE and OmcA protein families is presented. Finally, Chapter 6 explores the electrochemical properties of two multi-heme proteins from Nitrosomonas europaea: cytochrome c554 and hydroxylamine oxidoreductase (HAO). Cytochrome c554, a tetraheme cytochrome, has been shown to have cooperativity between two of its heme groups and gating has been. observed in protein film voltammetry (PFV) experiments. This gating is further explored in this Chapter. The enzymatic hydroxylamine reduction by HAO, a reverse reaction, is also presented.
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Rosenthal-Kim, Emily Quinn. "Green Polymer Chemistry: Synthesis of Poly(disulfide) Polymers and Networks." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1386525065.
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Utter, Bryan David. "PHEROMONE-INTERACTING REPLICATION PROTEIN CONTROLS ENTEROCOCCAL CONJUGATIVE PLASMID HOST RANGE AND STABILITY THROUGH DISULFIDE BONDS." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/211277.
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Microbiology and ImmunologyPh.D.
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.
Temple University--Theses
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Ogawa, Nozomi. "Resolving Disulfide Bond Patterns in SNAP25B Cysteine-Rich Region using LC Mass Spectrometry." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3651.
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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.
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Briggs, David Blaine. "BIOCHEMICAL CHARACTERIZATION OF ADIPONECTIN OLIGOMERIZATION." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/145741.
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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.
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Zhang, Yun. "Mass Spectrometric Analysis of Thiol Proteins/Peptides Following Selenamide Derivatization And Electrolytic Reduction of Disulfide Bonds." Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1347395762.
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Postma, Tobias Maria. "Novel Disulfide Formation Strategies in Peptide Synthesis." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/279370.
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Cysteine (Cys) is a unique amino acid due to its ability to form reversible covalent disulfide bonds. These bonds cause conformational constraints which make peptides or proteins more rigid and stable. In addition, the conformational constraints induced by disulfide bonds can result in highly potent molecules as illustrated by the vast number of disulfide-rich plant and animal peptide toxins. Furthermore, disulfide-rich peptides are gaining importance as pharmaceuticals and therefore powerful new synthetic strategies are needed. Disulfide-rich peptides can be prepared synthetically by several methods of which the state-of-the-art is described in chapter 1. However, most methods are time consuming or use harsh reagents. Herein, we describe the results of our research with the main goal of introducing and enriching novel molecular tools to aid in the preparation of disulfide-rich peptides.
In chapter 2 trimethoxyphenylthio (S-Tmp) is described as a novel Cys protecting group in Fmoc solid phase peptide synthesis replacing, the difficult to remove, tert-butylthio. S-Tmp and dimethoxyphenylthio (S-Dmp) were successfully used for Cys protection in a variety of peptides. Moreover, both groups can be removed in 5 min with mild reducing agents. S-Tmp is recommended for Cys protection as it yields crude peptides of high purity.
In chapter 3 N-chlorosuccinimide is described as a widely applicable on-resin disulfide forming reagent. Disulfide bond formation was completed within 15 minutes in DMF. This strategy was successfully used in the synthesis of oxytocin, and a regioselective synthesis of an α–conotoxin. Moreover, disulfide formation with N-chlorosuccinimide was found to be compatible with oxidation prone methionine and tryptophan.
In chapter 4 a novel method has been developed for the efficient formation of peptide disulfide bonds under aqueous conditions using N-chlorosuccinimide. Complete disulfide bond formation is achieved in 15 minutes with solvent mixtures containing water and acetonitrile. This method has applicability when on-resin disulfide formation gives poor results.
In chapter 5 a novel immobilized N-chlorosuccinimide resin was developed for peptide disulfide bond formation in combinatorial libraries. The resin is prepared in a simple two-step process from commercial starting materials. Disulfide formation is initiated by adding a peptide solution to the resin, and excess reagent is removed by a convenient filtration upon completion of disulfide formation. Completion of disulfide formation is rapid and clean, as demonstrated by the oxidation of a small nonapeptide library. This immobilized reagent allows a wider scope for the use of N-chlorosuccinimide based disulfide formation in combinatorial chemistry.
In chapter 5 we describe Cys pseudoproline-containing peptides with short deprotection times in TFA, contrary to other studies. The deprotection times fell in the same range as other protecting groups commonly used in SPPS. Moreover, a novel application for Cys pseudoprolines is disclosed in which this protecting group acts as a peptide macrocyclization-enhancing moiety that considerably reduces reaction time.
In summary, a novel Cys protecting group was introduced; N-chlorosuccinimide was found to be an efficient disulfide-forming reagent on-resin as in solution; an Immobilized derivative of N-chlorosuccinimide was described for combinatorial libraries; and Cys pseudoprolines with short deprotection times were observed which enhanced macrocyclization. These methods are a useful addition to the molecular tools available to prepare complex disulfide-rich peptides.La cisteína (Cys) es un aminoácido único debido a su capacidad para formar enlaces disulfuro que son reversibles. El capítulo 1 describe la gran variedad de métodos que existen para preparar este tipp de péptidos. La mayoría de éstos comportan protocolos tediosos y/o requieren reactivos agresivos. En esta misma parte, describimos los objetivos de nuestra investigación que ha estado enfocada a aumentar y a enriquecer el arsenal de herramientas químicas para preparar péptidos ricos en puentes disulfuro. . En el capítulo, el 2 trimetoxifeniltio (S-Tmp) se describe como un nuevo protector de la Cys compatible con la estrategia de síntesis de péptidos en fase sólida basada en el grupo Fmoc. El S-Tmp ha estado diseñado para sustituir el grupo terc-butiltio, que es difícil de eliminar. El grupo S-Tmp se puede eliminar en 5 min con agentes reductores suaves. Los crudos de los péptidos que se obtienen con el grupo S-Tmp son de gran pureza. En el capítulo 3, se describe la N-clorosuccinimida como un reactivo eficaz para la formación de enlaces disulfuro sobre resina. La formación de enlaces disulfuro tiene lugar en 15 minutos, utilizando DMF como disolvente. La validez de esta estratregia se ha demostrado mediante la sínntesis de varios péptidos. Este método es compatible con la presencia de metionina y de triptófano que son propensos a la oxidación. En el capítulo 4 se describe la utilización de la N-clorosuccinimida para la formación eficiente de enlaces disulfuro en condiciones acuosas. El proceso tiene lugar en 15 minutos . En el capítulo 5, se decribe una resina que lleva inmovilizada N-clorosuccinimida, que ha resultado ser muy conveniente para la formación de enlaces disulfuro en la preparación de bibliotecas combinatorias de péptidos. La formación del puente disulfuro es rápida y limpia. Este reactivo inmovilizado debe encontrar una gran utilización en química combinatoria . En el capítulo 6, se describe la utilización de pseudoprolinas derivadas de Cys en la síntesis de péptidos que contengan Cys. La formación de la Cys a partir de la pseudoprolina de Cys tiene lugar con tratamientos cortos de TFA. En este capítulo se ha descrito que las pseudoprolinas de Cys pueden favorecer las reacciones de macrociclación. En resumen, creemos que los métodos que presentamos son una aportación importante a los ya existentes para la preparación péptidos con puentes disulfuro.
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Dayalan, Saravanan, and saravanan dayalan@rmit edu au. "On the Structure Differences of Short Fragments and Amino Acids in Proteins with and without Disulfide Bonds." RMIT University. Computer Science and Information Technology, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081128.122615.
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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.
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MIZUOCHI, TSUGUO, MUNEHIRO NAKATA, IRVING BOIME, YUTAKA TOMODA, FUMITAKA KIKKAWA, MADOKA FURUHASHI, NOBUHIKO SUGANUMA, and TAKAYUKI MORIWAKI. "Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β -subunit by disruption of disulfide bonds." Thesis, Springer, 1997. http://hdl.handle.net/2237/16683.
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Atcher, Ubiergo Joan. "Disulfide-based dynamic combinatorial libraries of macrocyclic pseudopeptides as bio-inspired complex chemical systems." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/305491.
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Dynamic combinatorial chemistry proposes the creation of a library of compounds (dynamic combinatorial library, DCL) inter-connected through reversible chemical processes. The concentrations in a DCL are determined by differences in the free energy between the constituents and, therefore, the variations in the composition of the library contain valuable information about changes in the stability of the members. The adaptive nature of DCLs can be intimately correlated with a very simple expression of molecular evolution.
The main objective of this thesis is to use DCLs as bio-inspired complex chemical systems for the minimalistic experimental modeling of different processes of biological interest.
In Chapter 1, fourteen new pseudopeptidic dithiols were designed, synthesized and fully characterized in order to be used as bipodal building blocks (BBs). Their design is based on a C2-symmetric scaffold consisting of a central m-phenylenediamine chromophore that rigidly joins two identical arms, each formed by an amino acid with a mercaptoacetyl moiety attached to the N-terminus. The pseudopeptidic nature provides the BBs with peptide-like information, differently charged functional groups and chiral information. In Chapter 2, suitable experimental conditions were developed for the generation of disulfide-based DCLs from the mixture of the synthesized BBs. For this purpose, the use of DMSO as a co-solvent has beneficial effects in the thiol-disulfide dynamic covalent chemistry. Apart from the general gain in the aqueous solubility of the organic molecules, it promotes the thiol oxidation, highly reducing the reaction time for the disulfide formation. Besides, it accelerates the disulfide exchange, allowing the system to fully equilibrate even at slightly acidic pH.
In Chapter 3, a minimalistic DCL was used to reproduce adaptive trends described for the evolution of biological systems. Thus, the addition of salt to a dynamic library of macrocyclic pseudopeptides induces the amplification of those species concentrating anionic amino acids, with the Asp derivatives showing a better salt-adaptation than the Glu counterparts. Structural studies suggest a folded conformation for the amplified members and reveal the selection of those species showing a smaller accessible surface area. The adaptive process is driven by the increase of the ionic strength and has a remarkable resemblance with the natural evolution of the proteins of halophilic microorganisms for surviving in hypersaline media. In Chapter 4, the same external stimulus was studied in a larger DCL consisting of 21 differently charged dimeric macrocycles. The salt-induced adaptation of this complex system was characterized in a top-down fashion by the dynamic deconvolution into the minimal components.
Additionally, structural studies were performed for selected species. The salt-response of the members of the library can be classified in different families attending to the charges, and the behavior of each member is determined by a combination of its structural information and the co-adaptive relationships with the other members of the complex network.
Finally, in Chapter 5, a simple DCL consisting of homo- and heterochiral dimeric pseudopeptides was used to study how the chiral information is transmitted from the molecular to the macromolecular level. A decrease in the polarity of the medium induces a homochiral self-sorting process driven by polar intramolecular interactions. Additionally, the homochiral selectivity also increases with the temperature, indicating a positive entropic contribution. Preliminary NMR experiments suggest significantly different conformations for the homo- and heterochiral species.
Overall, the adaptive nature of DCLs, together with a suitable bio-inspired design, have demonstrated to allow the minimalistic experimental modeling of different processes of biological interest such as the natural evolution of the halophilic proteins, the co-adaptive relationships in a complex network and the homochiral self-sorting phenomenon.Les quimioteques dinàmiques (DCLs de “dynamic combinatorial libraries”) estan formades per una mescla de compostos interconnectats per mitjà de processos químics reversibles. Aquests sistemes dinàmics presenten la capacitat d’adaptar la seva composició a la presència d’un estímul extern. L’objectiu principal d’aquesta tesi és utilitzar DCLs com a sistemes químics complexos pel modelatge experimental de diversos processos d’interès biològic. En el Capítol 1 es van dissenyar, sintetitzar i caracteritzar catorze ditiols amb informació estructural de tipus peptídic, cadenes laterals amb diferents càrregues i informació quiral. A continuació, en el Capítol 2, es van establir unes condicions experimentals adequades per a generar DCLs de disulfurs a partir de l’oxidació dels ditiols sintetitzats. En aquest sentit, l’ús de DMSO com a codissolvent orgànic va demostrar tenir una sèrie d’efectes beneficiosos. Seguidament, en el Capítol 3, es va utilitzar una DCL minimalista de disseny bioinspirat per a reproduir tendències adaptatives pròpies de processos evolutius biològics. Així, es va observar que els canvis que provoca l’increment de la salinitat en la composició d’una DCL de pseudopèptids macrocíclics, tenen una notable similitud amb l’evolució natural de les proteïnes dels microorganismes halòfils. En el Capítol 4 es va estudiar l’efecte del mateix estímul extern en la composició d’una DCL complexa formada per espècies amb diferents càrregues. S’evidencià que el comportament de cada membre de la quimioteca està determinat per la seva informació estructural i per les múltiples relacions coadaptatives que aquest estableix amb la resta de membres de la xarxa molecular. Finalment, en el Capítol 5, es va utilitzar una DCL minimalista formada per espècies estereoisomèriques per estudiar el fenomen d’autoordenació homoquiral.
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Schmitz, Thomas [Verfasser]. "Influence of disulfide-bonds on structural and functional properties of peptides and proteins - Case studies on FXIIIa inhibitor tridegin and µ-conotoxin PIIIA / Thomas Schmitz." Bonn : Universitäts- und Landesbibliothek Bonn, 2021. http://d-nb.info/1235524582/34.
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King, Nadine Verfasser], Dieter [Akademischer Betreuer] [Langosch, Volker Gutachter] Bruß, and Dieter [Gutachter] [Langosch. "Mapping disulfide bonds and free sulfides in the small hepatitis B envelope protein S / Nadine King ; Gutachter: Volker Bruß, Dieter Langosch ; Betreuer: Dieter (Prof. Dr. Langosch." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1201819938/34.
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Cohen, Noah R. "Exploring the Complex Folding Free Energy Landscapes of a Series of β-rich Proteins." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1050.
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Protein aggregation is deleterious to human health and detrimental to therapeutic shelf-life. The physical processes that induce aggregation are the same processes that drive productive folding reactions. As such, protein aggregation is a non-productive form of protein folding. To gain insight into the steps that serve as a partition between the folding and aggregation reactions, the folding mechanisms of several β-rich proteins with links to human disease or medicine were examined.
In the ALS-linked protein, SOD1, a subpopulation of the unfolded ensemble is found to be a common source of both nonnative structure and frustrated folding. These behaviors are only observed upon the reduction of the intrinsic disulfide bond, indicating that this covalent interaction wards against aggregation. The nonnative structure presents an attractive target for the development of new therapeutic agents.
In VH domains from therapeutic mAbs, the intramolecular disulfide bond protects against aggregation. However, it can also introduce complexity to the folding mechanism. This complexity is linked to the formation of a strained orientation of the disulfide bond. This strained orientation of the disulfide in certain VH domains is energetically unfavorable enough to disrupt the formation of the disulfide in the full length mAbs. The novel relationship observed between disulfide orientation, folding complexity, and incomplete oxidation warrants further examination in other Ig domains.
Overall, these results demonstrate that mapping the folding free energy landscape for proteins with roles in human disease or therapeutics can provide valuable insights for developing and improving treatment options.
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Erlandsson, Lisa-Marie. "Understanding the Involvement of Leukocyte Cell-derived Chemotaxin 2 (LECT2) in Amyloidosis." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-162631.
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Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.
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Touchard, Axel. "Biodiversité, biochimie et pharmacologie des peptides de venins de fourmis." Thesis, Antilles-Guyane, 2015. http://www.theses.fr/2015AGUY0829/document.
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Les venins sont des armes sophistiquées, utilisées par les organismes venimeux pour se défendre des prédateurs, ainsi que pour paralyser et tuer leurs proies. Mais dans la nature, le bien n’est jamais très loin du mal, les toxines venimeuses pouvant se révéler être des agents thérapeutiques efficaces. Les peptides de venins de fourmis ont donc été étudiés dans cette thèse afin de déterminer le potentiel de ces toxines pour la découverte de molécules thérapeutiques innovantes. A l’instar des autres venins d’insectes, les venins de fourmis restent peu étudiés, principalement en raison de la petite taille de ces insectes et des quantités limitées de venins disponibles. Cependant, les fourmis offrent l’avantage d’être des insectes sociaux très abondants dans tous les milieux terrestres. En collectant les venins de plusieurs individus, il est donc possible d’obtenir des quantités suffisantes de venin pour les analyses biochimiques et pharmacologiques.Afin d’assurer la reproductibilité des analyses, une identification taxonomique correcte est nécessaire. Dans cette optique, un outil de chimiotaxonomie a été développé durant cette thèse (permettant ainsi de regrouper les venins provenant de plusieurs colonies afin de compenser les faibles quantités de matériel biologique par individu ou par colonie).Ensuite, nous nous sommes intéressés aux facteurs écologiques impliqués dans la diversification des venins de fourmis. Pour cela, la toxicité et la composition des venins de fourmis ont été analysés en relation avec le polyéthisme, la spécialisation alimentaire et la spécialisation défensive.La diversité écologique des fourmis a amplement contribuée à la diversification des venins. En étudiant les venins de 82 espèces de fourmis, nous avons révélé la grande diversité structurale des toxines. Bien que la majorité des peptidomes sont composés par de petits peptides linéaires, des peptides structurés par des ponts disulfure ont été révélés dans de nombreux venins et constituent de nouvelles familles structurales de toxines.La purification de certains de ces peptides à ponts disulfure a permis leur caractérisation biochimique et l’évaluation de leur rôle biologique. Ainsi nous avons décrit un groupe de peptides neurotoxiques, baptisés les formicitoxines qui sont capables de bloquer les canaux calcium humains de type L. La commutatoxine est, quant à elle, un peptide avec un pont disulfure qui semble activer les récepteurs humains TRPV1 et TRPV3 et laisse supposer une implication dans l’induction de la douleur chez les mammifères.La grande diversité des peptides mise en évidence dans les venins, associée à la grande diversité écologique et taxonomique des fourmis, suggère que les venins de fourmis constituent un nouveau champ d’exploration prometteur pour la recherche de molécules thérapeutiques et insecticides. Les venins de fourmis s’ajoutent à la chimiothèque conséquente déjà représentée par les venins des autres animaux venimeuxVenoms are sophisticated weapons employed by venomous organisms to ward off predators, as well as to subdue and kill prey. However, in nature, good is never far from bad and venom toxins may prove to be efficient therapeutic agents. Ant venom peptides were investigated in the course of this thesis to evaluate their potential in the discovery of novel drugs. Like other insect venoms, ant venoms remain understudied, mainly due to the small size of individual ants and, so, the limited a mount of venom available. The ecological diversity of ants has largely contributed to venom diversification. By studying the venom peptidomes from 82 ant species, we have revealed the great structural diversity of the toxins. Although the majority of the peptidomes are comprised of small and linear peptides, peptides structured by disulfide bonds were also brought to light in numerous venoms and constitute novel structural classes of toxins. The purification of some of these disulfided peptides permitted their biochemical characterization and the assessment oft heir biological functions. The enormous peptide diversity revealed among venoms combined with the great ecological and taxonomical diversity of ants suggests that ant venoms constitute a promising new source in the search for both novel drugs and insecticides. Ant venom augments the vast bioactive molecules library represented by venoms from other venomous animals
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Zhu, Hongxin. "Structure-Function Studies of Bovine Pancreatic Phospholipase A2: The Roles of N-Terminal Residues in The Interfacial Activation and The Roles of Disulfide Bonds in The Structure, Stability, and Catalytic Function Cloning, Expression, Purification... /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487931993466424.
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Nguyen, V. D. (Van Dat). "Mechanisms and applications of disulfide bond formation." Doctoral thesis, Oulun yliopisto, 2015. http://urn.fi/urn:isbn:9789526207254.
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Abstract About one-third of mammalian proteins are secreted proteins and membrane proteins. Most of these proteins contain disulfide bonds in their native state, covalent links formed between the thiol groups of cysteine residues. In many proteins, disulfide bonds play an essential role in folding, stabilizing structure and the function of the protein. Therefore, understanding the pathways of disulfide bond formation is crucial for a wide range of medical processes and therapies. Disulfide bond formation is catalyzed by the Protein Disulfide Isomerase (PDI) family. To date the mechanisms of the PDIs in disulfide bond formation and pathways for disulfide bond formation have not been fully characterized. Here the structure of the substrate binding b’x domain of human PDI was determined. The structure shows that the b' domain has a typical thioredoxin fold and that the x region can interact with the substrate binding site of the b' domain. Specifically, the x region of PDI can adopt alternative conformations during the functional cycle of PDI action and that these are linked to the ability of PDI to interact with folding substrates. In addition, this study showed that two human proteins, GPx7 and GPx8 are involved in disulfide bond formation. The addition of GPx7 or GPx8 to a folding protein along with PDI and peroxide allows the efficient oxidative refolding of a reduced denatured substrate protein. Finally, this thesis includes the development of a system for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli. It showed that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond, allows the formation of native disulfide bonds in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction. Introduction of Erv1p and a disulfide isomerase, e.g. PDI, allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. This system is able to express high levels of complex disulfide bonded eukaryotic proteinsTiivistelmä Noin kolmasosa kaikista nisäkkäiden proteiineista on solun ulkopuolelle eritettäviä proteiineja ja kalvoproteiineja. Monet näistä proteiineista sisältävät natiivissa konformaatiossaan disulfidisidoksia, jotka ovat kovalenttisia sidoksia kysteiinitähteiden tioliryhmien välillä. Useissa proteiineissa näillä disulfidisidoksilla on keskeinen rooli proteiinin laskostumisessa, kolmiulotteisen rakenteen stabiloinnissa sekä proteiinin toiminnassa. Disulfidisidosten muodostumisen taustalla olevien mekanismien tunteminen onkin tärkeää monien lääketieteellisten prosessien ja hoitomenetelmien kannalta. Disulfidisidosten muodostumista katalysoivat proteiinidisulfidi-isomeraasi (PDI) -perheeseen kuuluvat entsyymit. PDI entsyymien toimintamekanismeja ja disulfidisidosten muodostumisen reaktioreittejä ei kuitenkaan vielä tunneta tarkasti. Tässä väitöskirjassa selvitettiin ihmisen PDI entsyymin substraattia sitovan b’x alayksikön rakenne. Rakenteesta voidaan todeta b’ alayksikön laskostuminen tyypilliseen tioredoksiini muotoon sekä x alueen interaktio b’ alayksikön substraattia sitovan kohdan kanssa. PDI entsyymin katalysoiman reaktioketjun aikana x alayksikkö voi muuttaa konformaatiotaan mahdollistaen PDI entsyymin interaktion laskostuvien substraattiproteiinien kanssa. Tässä tutkimuksessa osoitettiin myös kahden ihmisen proteiinin, GPx7 ja GPx8 osallistuminen disulfidisidosten muodostumista katalysoiviin reaktioihin. GPx7 ja GPx8 entsyymien lisäys laskostumisreaktioon yhdessä PDI:n ja vetyperoksidin kanssa mahdollistaa pelkistetyn, denaturoidun substraattiproteiinin tehokkaan, hapettaviin reaktioihin perustuvan uudelleenlaskostumisen natiiviin muotoonsa. Osana tätä väitöstutkimusta kehitettiin menetelmä, joka mahdollistaa disulfideja sisältävien proteiinien tehokkaan tuoton E.colin solulimassa. Menetelmässä sulfhydryylioksidaasina ja FAD:sta riippuvana disulfidisidosten muodostumisen katalysaattorina toimiva Erv1p mahdollistaa disulfidisidosten muodostumisen E.colin solulimassa myös ilman solun pelkistävien reaktioreittien geneettistä poistamista. Erv1p yhdessä disulfidi-isomeraasin, kuten PDI, kanssa mahdollistaa oikein laskostuneiden, useita disulfidisidoksia sisältävien eukaryoottisten proteiinien tehokkaan tuotannon E.colin solulimassa. Menetelmällä pystytään tuottamaan suuria määriä monimutkaisia disulfidisidoksellisia proteiineja
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Bechtel, Tyler Jeffrey. "Chemical-Proteomic methods to interrogate disulfide-bond formation:." Thesis, Boston College, 2019. http://hdl.handle.net/2345/bc-ir:108613.
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Thesis advisor: Eranthie WeerapanaDisulfide-bonding cysteine residues perform critical roles in the structural stabilization and redox regulation of protein function. Secreted proteins are often enriched for structural disulfide bonds conferring conformational stability in the oxidizing extracellular environment. The controlled formation of disulfide bonds in secreted proteins is regulated in the endoplasmic reticulum (ER) by the protein disulfide isomerase (PDI) family. To investigate disulfide-bond formation in the ER, quantitative chemical-proteomic methods were coupled to subcellular-fractionation-based ER enrichment. Cysteine reactivity studies identified highly reactive post-translationally modified cysteine residues including disulfide-bonding cysteines. Upon discovering a highly reactive population of traditionally oxidized cysteines, the percentage of oxidation for cysteines localizing to the ER was determined. Next, ER function was chemically perturbed to evaluate changes to cysteine oxidation following upregulation of the unfolded protein response (UPR). Disulfide bond formation was specifically disrupted in the ER by CRISPR-Cas9-mediated PDIA1 and PDIA4 knockout. The effects of PDI knockout on cancer cell phenotype and changes to cysteine oxidation states were evaluated. Finally, in vitro studies were performed to evaluate PDIA4 oxidase activity and identify potential PDIA4-selective inhibitors. In the future, the platforms developed within may be applied to profiling changes to cysteine oxidation in other biological systems such as other organelles and disease states
Thesis (PhD) — Boston College, 2019
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Giribaldi, Julien. "Synthèse de peptides bioactifs inspirés des venins." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTS124.
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Les extraits naturels tels que les venins animaux constituent une source importante de peptides bioactifs à visée thérapeutique. Parmi les peptides dérivés de venins utilisés en médecine, citons l’Eptifibatide, un médicament antiplaquettaire développé à partir de l’echistatin provenant d’une vipère, le Ziconotide, un puissant analgésique identifié dans le venin d’un cône ou encore l’Exenatide, un agoniste du récepteur du glucagon-like peptide 1 isolé de la salive du monstre de Gila et utilisé pour le traitement du diabète de type 2. Ces peptides riches en ponts disulfures provenant des venins présentent une structure tridimensionnelle contrainte et une stabilité plasmatique accrue comparé aux peptides linéaires. La conservation et la ressemblance des récepteurs de la proie/du prédateur avec les récepteurs humains, fait des peptides de venins une source unique de composés tête de série pour le design d’outils pharmacologiques et de composés thérapeutiques. Il est estimé que moins de 1% des peptides de venins ont été caractérisé pharmacologiquement. Ce projet a pour objectif d’explorer la pharmacologie de nouveaux peptides isolés de venins en utilisant la synthèse peptidique en phase solide basée sur la chimie Fmoc (Fmoc-SPPS) ainsi que des stratégies de repliement oxydant et régiosélectif afin de produire le peptide correctement replié responsable de l’activité biologique pour sa caractérisation ultérieure. Alors que la première partie de ce projet est dédiée à la synthèse de peptides de venins linéaires et pauvre en pont disulfures, la seconde partie sera consacrée à la synthèse de peptides riches en pont disulfures via des stratégies de repliement oxydatif et régiosélectifs. Dans un dernier chapitre, nous allons explorer les approches de protéomique intégrées à la transcriptomique pour l’identification de nouvelles séquences à partir des venins. Dans l'ensemble, ce projet permettra de mieux comprendre la pharmacologie des peptides de venins et mènera à la conception de nouveaux outils pharmacologiques et de potentiels candidats médicamentsNatural extracts such as animal venoms are an important source of bioactive peptides for therapeutic purposes. Peptides derived from venoms currently used in medicine include Eptifibatide, an antiplatelet drug developed from echistatin, a toxin isolated from a viper, Ziconotide, a potent analgesic identified in the venom of a cone snail and Exenatide , a glucagon-like peptide 1 receptor agonist isolated from the saliva of the Gila monster and used for the treatment of type 2 diabetes. These disulfide-rich venom peptides exhibit a constrained three-dimensional structure and increased plasma stability compared to linear peptides. Conservation of prey / predator receptors with human receptors makes venom peptides a unique source of lead compounds for the design of pharmacological tools and therapeutic compounds. It is estimated that less than 1% of the venom peptides have been pharmacologically characterized. Thus, this project aims to explore the pharmacology of novel venom-isolated peptides using solid phase peptide synthesis based on Fmoc chemistry (Fmoc-SPPS) as well as oxidative and regioselective folding strategies to produce the correctly folded and biologically active peptide for subsequent characterization. While the first part of this project is dedicated to the synthesis of linear and disulfide-poor venom peptides, the second part will be dedicated to the synthesis of disulfide-rich peptides via oxidative and regioselective folding strategies. Finally, we will use proteomic approaches integrated with transcriptomic data for the identification of new sequences from venoms. Overall, this project provides a better understanding of the pharmacology of venom peptides and identifies leads for the development of new pharmacological tools and potential drug candidates
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Love, Katie. "Investigating the Role of Disulfide Bond Formation in FABP5." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:107265.
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Thesis advisor: Abhishek ChatterjeeThesis advisor: Eranthie Weerapana
EGF signaling activates multiple pathways within the cell that lead towards proliferation, rendering this pathway of interest for cancer therapy. Recent studies focused on triple-negative breast cancer have shown that EGF-induced tumorigenesis strongly correlates with the up-regulation of FABP5, which shuttles fatty acids from the cytoplasm of cells to the nucleus. Our work began with the identification of redox active cysteine residues upon EGF activation in situ using a caged electrophile to perform live cell labeling. In these studies, the C120 residue of FABP5 was identified as a cysteine with high redox activity and thus became a subject of further interest. The characterization of redox active cysteine residues yields important information about protein structure and function. We have confirmed these results via in-gel fluorescence and developed fluorescence assays to probe the significance of C120 and C127 in FABP5. Two fatty acids were chosen based on their conformation in the FABP5 binding pocket. Upon the addition of a fatty acid, wild type protein showed a decrease in fluorescence indicating that the fatty acids were outcompeting the fluorophores used. Future studies will investigate both wild type and mutant versions of FABP5 with emphasis on determining potential disulfide bond formation via phosphoproteomics and western blotting techniques
Thesis (MS) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Kouwen, Thijs R. H. M. "Protein secretion and disulfide bond handling in bacillus subtilis." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2009. http://irs.ub.rug.nl/ppn/315686960.
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Bessette, Paul Henry. "Engineering and physiology of disulfide bond isomerization in Escherichia coli /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p9995165.
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Sumbalová, Lenka. "Bioinformatický nástroj pro návrh disulfidických můstků v proteinové struktuře." Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2016. http://www.nusl.cz/ntk/nusl-255430.
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Proteins are substances with great usage. For industrial usage, proteins are often taken from their natural enviroment. In foreign environment, it proteins can unfold and their function can be compromised. This is the reason for stabilization of proteins and one of ways to stabilization is using disulphide bonds. This work describes basic terms related to protein stabilization - proteins, their structure and interactions within them, basic terms from thermodynamics. Problem of protein stability is discussed and the factors which stabilize or destabilize protein are enumerated with the emphasis on disulphide bonds. Existing approaches to disulphide bonds design, dataset for testing own tool are described. Implementation of the tool using geometrical properties of the bonds and fl exibility of places in protein is described. The tool was tested on proteins with native disulfide bonds and compared to existing tools, also metrics FRO (fractional rank order) was used. Native disulfide bond was found in 64 % of cases, in 60 % of cases this native disulfi de bond was in the first quarter of ordered found disulfi de bonds.
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Matsumiya, Nozomi. "Optimization of disulfide mapping using mass spectrometry." Thesis, Kansas State University, 2009. http://hdl.handle.net/2097/1358.
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Master of ScienceBiochemistry
John Tomich
One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.
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Qiu, Ji. "Genetic and biochemical analysis of disulfide bond isomerization in Escherichia coli /." Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3008423.
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Jain, Surbhi. "Role of Disulfide Bond Rearrangement in Newcastle Disease Virus Entry: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/379.
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Newcastle disease virus (NDV), an avian paramyxovirus, enters the host cell by fusion of viral and host cell membranes. The fusion of two membranes is mediated by the viral fusion (F) protein. The F protein, like other class I fusion proteins, is thought to undergo major conformational changes during the fusion process. The exact mechanism that leads to major refolding of F protein is not clear. Recently, it has been proposed that disulfide bond reduction in the fusion protein of some viruses may be involved in the conformational changes in fusion proteins. In some viruses, the reduction of disulfide bonds in the fusion protein is mediated by host cell disulfide isomerases belonging to the protein disulfide isomerase (PDI) family. In this study, the role of disulfide bond isomerization in the entry of NDV was analyzed. Using inhibitors of thiol-disulfide isomerases, we found that blocking the reduction of disulfide bonds in the fusion protein inhibited cell-cell fusion as well as virus entry into the host cell. Also, over-expression of isomerases belonging to the PDI family significantly enhanced cell-cell fusion. Taken together, these results suggest that free thiols play an important role in fusion mediated by NDV glycoproteins.
Using a thiol specific, membrane impermeable biotin, MPB, we found that free thiols are produced in cell surface-expressed NDV F protein. The production of free thiols was inhibited by inhibitors of thiol-disulfide isomerases. Over-expression of isomerases belonging to the PDI family enhanced detection of free thiols in F protein. In F protein, present in virions or in virus-like particles, free thiols were detected only after the particles were attached to target cells. Taken together, these results suggest that free thiols are produced in F protein and the production of free thiols is mediated by host cell thiol-disulfide isomerases.
Using conformation sensitive antibodies, we also studied the conformation of cell surface-expressed F protein in the presence ofthiol-disulfide isomerase inhibitors or in cells over-expressing thiol-disulfide isomerases. In the presence of thiol-disulfide isomerase inhibitors, the cell surface-expressed F protein was in a prefusion conformation while in cells over-expressing thiol-disulfide isomerases the F protein was in a post-fusion conformation.
We also correlated the production of free thiols to the conformational changes in F protein. Using temperature-arrested intermediates or F protein with mutations in heptad repeat domains, which are defective in attaining intermediate conformations, we found that free thiols are produced before any of the proposed conformational changes in F protein. Also, the production of free thiols in F protein was found to be independent of its activation by hemagglutinin-neuraminidase (HN) protein. These results suggest that free thiols are probably required for the activation of F protein during membrane fusion.
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Mimura, Hisatoshi, Yoichi Nakanishi, Masayoshi Maeshima, and 正義 前島. "Oligomerization of H+-pyrophosphatase and its structural and functional consequences." Elsevier, 2005. http://hdl.handle.net/2237/6661.
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Zhao, Liang. "Post-translational modifications of SEL24K from salmon eggs and ZPA from Xenopus laevis eggs." Scholarly Commons, 2011. https://scholarlycommons.pacific.edu/uop_etds/160.
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Post-translational modifications (PTMs) of proteins play significant roles in regulation of biological activities and signal transduction. Examining their diversity is critical for understanding the mechanisms of cellular regulations. Among the various techniques employed for identification of PTMs, mass spectrometry has become a more and more important tool for detecting and mapping these covalent modifications and quantifying their changes. The two projects described in this dissertation focus mainly on the method development for characterization of two major PTMs, disulfide bonds and glycosylation. In the first project, the disulfide bond pattern of a rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha was assigned unambiguously based on a multi-enzyme digestion strategy in combination with MALDI-TOF mass spectrometry analysis. The disulfide bond pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. More importantly, an interesting phenomenon of gas-phase scrambling of disulfide bonds was observed during MALDI mass spectrometry analysis and a possible mechanism for this surprising scrambling was proposed. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. In the second project, the glycosylation of a glycoprotein ZPA from the vitelline envelope of Xenopus laevis was determined by applying a strategy of general proteolysis coupled with mass spectrometry. The vitelline envelope glycoproteins were first separated through SDS-PAGE. A nonspecific in-gel pronase digestion was performed on the excised band of ZPA to produce informative small glycopeptides. Lectin affinity chromatography was used for the enrichment of these glycopeptides. An in-gel PNGase F digestion was also carried out to release the N-linked glycans from ZPA. The enriched glycopeptides and glycans were finally analyzed by MS and MS/MS techniques on MALDI-TOF and MALDI-TOF/TOF instruments.
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Zhao, Yi. "Degradable molecularly imprinted polymers-synthetic antibody mimics for the vectorization of active molecules." Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2189.
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Les polymères à empreintes moléculaires (MIP) sont des matériaux synthétiques capables de mimer les anticorps biologiques. En effet, ils possèdent deux des principales caractéristiques de ces derniers, à savoir : la capacité de reconnaître et de se lier spécifiquement à des molécules cibles. De plus, leur synthèse facile, leur bas coût de production, leur haute spécificité et stabilité par rapport aux anticorps naturels font des MIP une alternative intéressante. En effet, les propriétés de reconnaissance moléculaire des MIP permettent d'envisager leur utilisation dans une vaste gamme d’applications. Ils sont ainsi largement exploités dans les sciences séparatives pour l'analyse d'échantillons environnementaux ou agro-alimentaires, ou comme élément de reconnaissance dans des biocapteurs. Récemment, des applications de ces matériaux dans les domaines biologiques et biomédicaux ont émergé comme pour la détection, l'extraction et l"élimination de molécules indésirables dans l'organisme, la vectorisation ou l'administration contrôlée des médicaments. Dans nos recherches, nous avons développé des MIP dégradables par voie biochimique ou enzymatique, ayant une application potentielle en tant que système de libération contrôlé de molécules. En général, les MIPs sont synthétisés par polymérisation radicalaire libre en utilisant une formulation composée de monomères fonctionnels, d'agents de réticulation, et d'une molécule cible servant à réaliser l'empreinte moléculaire. Dans ce travail de thèse, nous avons utilisé pour la synthèse de MIP dégradable des agents de réticulation clivables contenant, soit une fonction chimique dégradable par voie chimique ou enzymatique (ponts disulfures et phosphatediester), soit un disaccharide issus d'agro-ressources et pouvant être naturellement hydrolysé par des enzymes. En présence d'un réactif spécifique (agent réducteur ou enzyme), les liaisons dites "sensibles" aux réactifs chimiques ou enzymatiques peuvent être clivées, ce qui entraîne une dégradation de la matrice polymérique. Le polymère perdra alors sa capacité de reconnaissance et de liaison à la molécule cible et permettra la libération de celle-ci. Nous pensons donc, que les nouveaux MIP dégradables pourraient avoir un énorme potentiel comme vecteurs "intelligents" dans des applications médicales tels que les systèmes de libération contrôlée de médicament. Finalement, nous avons étudié la dégradation par des microorganismes de la structure de base de ce type de polymères, en utilisant comme modèles des chaines linéaires et réticuléesMolecularly imprinted polymers (MIPs) are biomimetic synthetic receptors that possess two of the most important features of biological antibodies – the ability to recognize and bind specific target molecules. Owing to their easier preparation, lower cost, higher specifity and stability compared to antibodies, they have the potential to be widely applied for environemental and food analysis. Recently, MIPs also emerged in the biochemical field as diagnostic tools, chemicals traps to remove undesirable substance from the body, or drug delivery systems, where usually the combination of biocompatibility and degradability after its use is desirable. Here, we developed biochemically or enzymatically degradable MIPs, which have potential applications as activation-modulated drug delivery systems. In general, MIPs are prepared by radical polymerization of functional monomers and cross-linkers in the presence of a target molecule acting as template. Degradable MIPs were synthesized using cleavable cross-linkers containing a degradable group (disulfide bond or phosphate ester bond) or derived from a natural disaccharide. In the presence of a cleaving reagent (reducing agent or enzyme), the chemo or enzyme-sensitive bond could be cleaved, resulting in the degradation of the polymer matrix. The degraded polymers looses the binding sites structure resulting in the loss of recognition and binding capacity towards the target molecules, and thus in the release of bound molecules. These degradable MIPs provide new opportunities as “smart” vectors for controlled delivery of active molecules in biomedical applications. Finally, the biodegradation of the polymer backbone by bacteria was investigated
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Prescesky, Elizabeth Joy. "The use of 2-D PAGE to detect disulfide bond-containing proteins in Pseudomonas aeruginosa." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq52939.pdf.
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Mills, Jamie E. "Structural investigation and allosteric properties of a unique disulfide bond in C-terminal Src kinase." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3320620.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed September 24, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 110-118).
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Kojukhov, Artyom. "Assessment of disulfide bond formation during co-translational folding of synonymous codon variants of recombinant gamma-B crystallin." Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu152571031230488.
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Pirneskoski, A. (Annamari). "The significance of the domains of protein disulfide isomerase for the different functions of the protein." Doctoral thesis, University of Oulu, 2003. http://urn.fi/urn:isbn:9514271726.
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Abstract
Protein disulfide bonds are covalent links formed between the thiol groups of cysteine residues. In many proteins, they have an important role in stabilizing the three-dimensional conformation of the polypeptide chain. Usually proteins are physiologically active and functional only when they are correctly folded. Protein folding takes place very soon after the synthesis of a new polypeptide chain. Proteins which are to be secreted from the cell fold in a specialized compartment, the endoplasmic reticulum (ER).
Folding and disulfide bond formation in the ER does not happen spontaneously, there are proteins which are specialized in assisting in these processes. Protein disulfide isomerase (PDI) is a multifunctional protein, which is capable of catalysing both of disulfide bond formation and folding of a protein. In addition, it has other functions: it is an essential part of two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein.
C-P4H is an enzyme essential in the formation of collagens, proteins found in connective tissue. The function of C-P4H is to catalyse the hydroxylation of prolines, which is essential for the structural stability of collagens. C-P4H is a tetramer, formed of two catalytic α subunits and two β subunits, which are identical to PDI. The function of PDI in C-P4H is apparently to keep it in a soluble, functionally active conformation.
In mammals there are several proteins similar to PDI, together forming a PDI family of proteins. They share both structural and functional similarities. One of these proteins is ERp57. It is specialized in assisting in the folding and disulfide bond formation of glycoproteins.
PDI consists of four domains, two of which contain a catalytic site for disulfide bond formation. One domain is the main site of interaction with other proteins and one domain is of unknown function. In this study, the role of these domains in the activities of PDI was investigated. The peptide-binding domain was characterized in detail. In addition, structural similarities of PDI and ERp57 were studied by formation of hybrid proteins containing domains of both and comparing the activities of these recombinant proteins to those of PDI.
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Takahashi, Yoh-Hei. "Biochemical analyses of the quinone-coupled enzyme, DsbB, of Escherichia coli involved in disulfide bond generation." 京都大学 (Kyoto University), 2007. http://hdl.handle.net/2433/136787.
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Mishra, Avdesh. "Effective Statistical Energy Function Based Protein Un/Structure Prediction." ScholarWorks@UNO, 2019. https://scholarworks.uno.edu/td/2674.
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Proteins are an important component of living organisms, composed of one or more polypeptide chains, each containing hundreds or even thousands of amino acids of 20 standard types. The structure of a protein from the sequence determines crucial functions of proteins such as initiating metabolic reactions, DNA replication, cell signaling, and transporting molecules. In the past, proteins were considered to always have a well-defined stable shape (structured proteins), however, it has recently been shown that there exist intrinsically disordered proteins (IDPs), which lack a fixed or ordered 3D structure, have dynamic characteristics and therefore, exist in multiple states. Based on this, we extend the mapping of protein sequence not only to a fixed stable structure but also to an ensemble of protein conformations, which help us explain the complex interaction within a cell that was otherwise obscured. The objective of this dissertation is to develop effective ab initio methods and tools for protein un/structure prediction by developing effective statistical energy function, conformational search method, and disulfide connectivity patterns predictor.
The key outcomes of this dissertation research are: i) a sequence and structure-based energy function for structured proteins that includes energetic terms extracted from hydrophobic-hydrophilic properties, accessible surface area, torsion angles, and ubiquitously computed dihedral angles uPhi and uPsi, ii) an ab initio protein structure predictor that combines optimal energy function derived from sequence and structure-based properties of proteins and an effective conformational search method which includes angular rotation and segment translation strategies, iii) an SVM with RBF kernel-based framework to predict disulfide connectivity pattern, iv) a hydrophobic-hydrophilic property based energy function for unstructured proteins, and v) an ab initio conformational ensemble generator that combines energy function and conformational search method for unstructured proteins which can help understand the biological systems involving IDPs and assist in rational drugs design to cure critical diseases such as cancer or cardiovascular diseases caused by challenging states of IDPs.
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Machado, Luciana E. S. F., Tun-Li Shen, Rebecca Page, and Wolfgang Peti. "The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation." AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2017. http://hdl.handle.net/10150/624478.
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The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.
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Silvennoinen, L. (Laura). "ERp57—Characterization of its domains and determination of solution structures of the catalytic domains." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514280547.
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Abstract
The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER.
Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP).
The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57.
Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).
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Kayatekin, Can. "The Coupling Between Folding, Zinc Binding, and Disulfide Bond Status of Human Cu, Zn Superoxide Dismutase: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/515.
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Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation.
In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded.
Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.
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Ogawa, Nozomi. "Detection of cellular redox status by transient receptor potential channels." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215577.
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Mimura, Hisatoshi, Yoichi Nakanishi, Masayoshi Maeshima, and 正義 前島. "Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure." Elsevier, 2005. http://hdl.handle.net/2237/6662.
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Chintalacharuvu, Koteswara Rao. "Disulfide bond formation between dimeric immunoglobulin A and the polymeric immunoglobulin receptor in cultured epithelial cells and rat liver." Case Western Reserve University School of Graduate Studies / OhioLINK, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=case1055268413.
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Snyder, Christopher H. "Effects of Target Protein Peptides and MLCK on Calcium Exchange With Calmodulin and the Disulfide-Bond-Locked Mutant Calmodulin's /." The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487935125880357.
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Vincent-Sealy, Lois V. "Investigation of the role of disulfide bond formation in the secretion and activity of virulence factors in Erwinia carotovora subspecies carotovora." Thesis, University of Warwick, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263819.
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Mély, Yves. "Proprietes de la proteine s100b : purification d'isoproteines, formation de ponts disulfure, etude d'un derive disulfure mixte." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13164.
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Tsiamantas, Christos. "Synthesis and structure-stability relationship of aromatic helical foldamers." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0029/document.
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Au niveau moléculaire, les fonctions des motifs hélicoïdaux sont souvent associées à la stabilité de cesarchitectures. Par exemple, lorsqu’une hélice α est isolée de la structure tertiaire des protéines, celle-cidevient alors flexible et perd son activité. Afin de contrôler la rigidité de ces architectures, différentesapproches ont été proposées dont la construction d’édifices moléculaires repliés de façon contrôlée : lesfoldamères. Notre équipe s’intéresse aux foldamères d’oligoamide aromatique hélicoïdaux et à l’heureactuelle plusieurs études ont déjà été menées afin de comprendre les facteurs influant la stabilité de telleshélices: la longueur de l’oligomère, le solvant et l’effet de l’introduction d’un espaceur aliphatique dans laséquence. Lors de ce travail nous nous sommes tout d’abord intéressés à la capacité de repliement de cinqmonomères aromatiques couramment utilisés pour la préparation de foldamères. Leur contribution dans lastabilité hélicoïdale du système a été évaluée par RMN, diffraction des rayons-X et HPLC chirale dynamique.Inspirés par le rôle des ponts disulfure dans les protéines, nous avons décidé d’explorer l’effet d’une telleliaison sur la stabilité des hélices d’oligoamide aromatique. Deux composés contenant une liaison disulfureintramoléculaire ou deux liaisons intermoléculaires ont été synthétisés et leur stabilité étudiée par RMN, HPLCChiral et Dichroïsme circulaire.Enfin, la synthèse d’hélices moléculaires de grandes dimensions (assimilables à celles de polymèresmonodisperses) a été réalisée par une approche convergente de triplement de longueur de segment via laformation de liaisons anhydrides entre blocs oligomériquesAt the molecular level, the functions of helical patterns are often directly associated with the stability of thesearchitectures, (in α-helices). For example, upon removal of such an entity from the protein’s tertiary structure,the peptidic helix becomes flexible and thus inactive. In order to control the rigidity of these architectures,several strategies have been used and the construction of completely artificial well folded molecules known asfoldamers is one them. Our group mainly focuses on helical aromatic oligoamide foldamers and to dateseveral studies have been carried out to investigate factors affecting the helical stability; the influence ofoligomer length, solvent effects and the effect of aliphatic linkers within a helical aromatic sequence.In the present study we investigate the helical propensity of five commonly used aromatic monomers infoldamer synthesis and by using NMR spectroscopy, X-ray crystallography and dynamic chiral HPLC weevaluate their contribution in helical stability. Additionally, inspired by the role of disulfide bonds in proteins wedecided to explore their effect on helical stability. For this reason intra- and inter-molecularly disulfide bondedcompounds were designed and synthesized. Their stability was studied using NMR spectroscopy, chiral HPLCand CD experiments.Finally, the synthesis of mono-disperse helical strings of polymeric dimensions through a convergent, segmenttripling strategy has been developed. This protection/deprotection free synthesis was carried out byconnecting oligomeric blocks via a labile anhydride functionality