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

The work presented in this thesis focusses primarily on the determination of protein structure at atomic resolution, with NMR spectroscopy as the principle investigative tool. The thesis is divided into four parts. Part I consists of Chapter 1 which provides an introduction to protein structure, folding and NMR spectroscopy. Part II, consisting of Chapters 2 and 3, describes the effects of aromatic interactions on nucleating structure in disordered regions of proteins, using variants of apo-cytochrome b5 as a model system. Part III consists of Chapter 4, which describes structural effects of introducing cross-strand disulfide bonds using variants of Thioredoxin. Part IV of this thesis consists of the Appendices A, B and C. Appendix A describes the purification and characterization of ilvM, the regulatory subunit of the E.coli enzyme AHAS II. Appendices B and C contain chemical shift information corresponding to Chapter 3 and Chapter 4 respectively. Part I : Introduction to protein structure, folding and solution structure studies Chapter 1 first gives a brief overview of protein structure followed by an introduction to protein folding, focussing on the forces involved in determining the final three-dimensional shape of the protein as well as the experimental and computational techniques involved in studying or predicting the fold of a given protein. The second section of this chapter details the methodology followed to obtain solution structures of proteins using NMR spectroscopy. Part II : Engineering aromatic interactions to nucleate folding in intrinsically disordered regions of proteins Chapter 2 describes site-specific mutagenesis, recombinant over-expression, purifica-tion and preliminary biophysical characterization of two aromatic mutants of the molten globule apo-cytochrome b5 (apocytb5) : H43F H67F cytochrome b5 (FFcytb5) and H43W H67F cytochrome b5 (WFcytb5). Analysis of the structure of wild-type apo - cytochrome b5 was done to introduce surface mutations and avoid perturbation of the interior pack-ing of the protein. The bacterial host E.coli BL21(DE3) was used for recombinant over-expression, and both mutant proteins were purified by anion-exchange chromatography followed by size-exclusion chromatography. Biophysical studies show a decrease in the hydrodynamic radii and surface hydropho-bicity of FFcytb5 and WFcytb5 compared to wt -apo cytb5. An increase in protein stability was also seen from the wt apocytb5 to WFcytb5 and FFcytb5 in the presence of the chemical denaturant Urea. Proton 1D NMR spectra exhibited sharp lines and good spectral dispersion in the amide region, indicating that both mutant proteins are well folded. In addition, conservation of two distinctive up field and downfield shifted resonances present in apocytb5 indicated that structural changes upon mutation accrued on the upon the scaffold of apocytb5. Chapter 3 describes solution structure studies to determine secondary and tertiary structure of FFcytb5 and WFcytb5. Structural studies were carried out using homonu-clear and heteronuclear NMR methods, for which isotopically enriched 15N- and 13C, 15N samples were prepared for each protein. Additionally a 2H, 13C, 15N ILV methyl labeled sample was prepared for FFcytb5 to obtain unambiguous NOE correlation data. The hydrogen bond network for WFcytb5 was determined using hydrogen/deuterium exchange data. The restraints required to define the orientations and interactions of the aromatic groups were obtained from 15N-edited NOESY HSQC, 13C -edited NOESY HSQC and 2D 1H - 1H NOE spectra. These correlations were crucial in determining the aromatic interactions present within each protein. The structure of FFcytb5 was calculated using 1163 NOE distance restraints, 179 φ and ψ dihedral angle restraints, along with 40 hydrogen bond restraints. Similarly the structure of WFcytb5 was calculated using 1282 NOE distance restraints, 177 φ and ψ dihedral angle restraints and 40 hydrogen bond restraints. The ensemble of structures obtained for FFcytb5 showed a root mean square deviation of 1.01±0.21 Å . The ensemble of structures obtained for WFcytb5 showed a root mean square deviation of 0.58±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were found to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The final tertiary structure of both FFcytb5 and WFcytb5 consisted of a mixed four strand β -sheet with a four helix bundle resting on top and were seen to align well, with an RMSD of 0.6 Å. A comparison of the solution structures of apocytb5 with FFcytb5 and WFcytb5 convincingly showed the nucleation secondary and tertiary structure well beyond the site of mutation. The presence of aromatic trimers, non-canonical in context of the wt apoc-ytb5, was confirmed upon analysis of the structures of FFcytb5 and WFcytb5, with NOE correlations assigned to verify these interactions. The reduction in the hydrodynamic radii of FFcytb5 and WFcytb5 in relation to apocytb5 was also verified from tsuperscript15N-NMR relaxometry studies. The nucleation of long-range structure using aromatic interactions has been demonstrated in proteins for the first time, and can in principle be used to incorporate aromatic residues and interactions in protein design. Structural data, chemical shift data and restraints lists used for structure calculation of WFcytb5 and FFcytb5 were deposited with the PDB (accession numbers 5XE4 and 5XEE) and BMRB(accession numbers 36070, 36071) respectively1. Part III : Structural consequences of introducing disulfide bonds into β - sheets Chapter 3 describes the solution structure studies on two mutants of E.coli Thiore-doxin which were designed to incorporate a disulfide bond between two anti-parallel β-strands at the edge of the β-sheet. One mutant was designed with a disulfide bond at the hydrogen bonding position (HB, 78c90cTrx) and the other with the disulfide bond at the non-hydrogen bonding position (NHB, 77c91cTrx). Here we study the structural changes that accompany the introduction of a cross-strand disulfide and whether such structural changes could be correlated with the previously seen thermodynamic and catalytic changes. Solution structure studies were conducted using a suite of multidimensional heteronu-clear NMR experiments, for which isotopically enriched 15N and 13C, 15N labelled samples were used. The solution structure for 77c91cTrx was calculated using 1190 NOE distance restraints, 199 φ and ψ dihedral angle restraints and 48 hydrogen bond restraints. The solution structure for 78c90cTrx was calculated using 1123 NOE distance restraints, 197 φ and ψ dihedral angle restraints and 50 hydrogen bond restraints. The ensemble of structures for 77c91cTrx showed an RMSD of 0.78± 0.13 Å while the RMSD for the ensemble of structures of 78c90cTrx was seen to be 0.76±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were seen to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The tertiary structures of both proteins were seen to have a 5-strand mixed β-sheet and 4 helices surrounding it. . A comparison of the solution structures of mutant and wt -Trx showed significant changes in secondary and tertiary structure. For example, an α helix was reduced from 3 turns to a single turn, and of the β-strands containing the mutation was elongated by 3 residues. A ≈ 50% loss of hydrogen bonds, primarily from the β -sheet, was seen for both mutants. The secondary and tertiary structure for both 77c91cTrx and 78c90cTrx was seen to be near identical, despite the greater strain of the disulfide bond at the hydrogen bonding position. In addition to this, the Ile75-Pro76 peptide bond is now seen to be in the trans conformation in 78c90cTrx, while in wt -Trx the Ile75-Pro76 peptide bond is in the cis conformation. This cis peptide bond is known to play a role in both folding and catalysis, and the solution structures were analyzed in the context of observed changes in folding and catalysis. The study shows that introducing disulfide bonds even at the edge of β sheets have long-range structural effects, and the structural effects cannot be directly correlated with the changes in stability. Part III: Appendix Appendix A describes the expression, purification and preliminary characterization of ilvM, the regulatory subunit of E.coliAHAS II, one of three enzyme isomers that catal-yse the first step in the synthesis of all branched chain amino acids. AHAS II is known to be insensitive to feedback regulation, but our studies showed that the presence of Ile, Leu and Val causes structural changes and increases the stability of ilvM. However we were not able to purify ilvM in sufficient quantities to proceed with solution structure studies. Appendices B and C contain chemical shift information for the structural studies carried out on FFcytb5, WFcytb5, 77c91cTrx and 78c90cTrx.

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.

No description available.

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.

DCL-1 (CD302) is a type I transmembrane C-type lectin receptor, which is expressed on monocytes, macrophages, granulocytes and dendritic cells. However, its extracellular domain lacks the amino acids motives essential for carbohydrate binding in the presence of calcium ions, suggesting that it does not have the classic binding capacity found in other C-type lectin receptors such as the mannose receptor. No exogenous or endogenous ligands have been identified yet, though. Due to internal colocalization with F-actin we can assume, that this unconventional lectin receptor plays a role not only in endocytosis and phagocytosis but also in the cell adhesion and migration. The receptor DCL-1 was first identified as a genetic fusion partner of human DEC-205 multilectin receptor in Hodgkin's lymphoma cell lines. The experimental part of this thesis deals with the characterization of disulfide bonds and data acquisition for validation of DCL-1 crystal structure. First the production and refolding conditions were optimized to obtain the highest amount of DCL-1 protein, precisely its extracellular domain. These optimal conditions were used to prepare the protein for in-gel digestion using specific endopeptidases in the presence of cystamine followed by LC-MS analysis. DCL-1 disulfide bonds were determined by comparing...

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 venimeux. / Venoms 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.

NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.

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.

No description available.