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4 Abstract Purinergic P2X receptors are membrane ion channels activated by extracellular ATP. There are seven isoforms of mammalian P2X receptors designated as P2X1-7, which according to their structure represent a specific family of ligand gated ionic channels, with extraordinary structural/functional properties. The P2X receptor consists of three subunits and each subunit has two transmembrane domains. Crystalographic data demonstrate that ionic channel pore is situated between the second transmembrane domains. Crystal structure of P2X4 receptor from the zebrafish (Danio rerio) is available in both open and closed state of the channel and the exact structure of ATP binding site is solved. The aim of this thesis was to study the structure-function relationships in a model of recombinant P2X4 receptor of the rat. By employing the point mutagenesis and electrophysiological recording, the functional importance of conserved cysteine residues in the ectodomain and amino acid residues which form the extracellular vestibule was investigated. All ten cysteins were substituted one by one with alanine or threonine and ATP-induced currents were measured from HEK293T cells expressing wild type (WT) and mutated P2X4 receptors. The results indicate that C116A, C126A, C149A and C165A mutations disrupt two disulfide bonds...

<p>The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in <em>Escherichia coli</em>, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys₂₇₈-Cys₄₄₃being the most important and Cys₂₁₆-Cys₄₂₀ and Cys₃₃₀-Cys₃₈₀ being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.</p>

The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in Escherichia coli, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys278-Cys443 being the most important and Cys216-Cys420 and Cys330-Cys380 being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.

N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...

Die vorliegende Arbeit beschäftigt sich mit der genetischen Optimierung der Spalthefe S. pombe für die biotechnologische Produktion von Fremdproteinen. Hierbei werden vor allem zwei Aspekte näher untersucht, zum einen die Stabilität des zu produzierenden Proteins und zum anderen die Bildung von Disulfidbrücken. Von anderen Organismen ist bekannt, dass die N-terminale AS im Verbund mit einem Lysinrest ein Protein destabilisieren kann. Das Modellprotein vVEGF besitzt an Position 2 einen Lysinrest (K2) und damit ein Hauptmerkmal eines derartigen Destabilisierungselementes. Falls das Protein dem Ubiquitin-vermittelten Abbau unterliegt, ist es wahrscheinlich, dass K2 eine essenzielle Rolle für die Stabilität dieses Proteins spielt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass K2 in S. cerevisiae destabilisierend wirkt, während es in S. pombe keinen destabilisierenden Effekt hat. Dieses Ergebnis spricht dafür, dass es Unterschiede im Ubiquitin-vermittelten Abbau von Proteinen in diesen beiden Hefen gibt. Der Schwerpunkt dieser Arbeit lag auf der Analyse und Optimierung der Bildung von Disulfidbrücken in S. pombe. Disulfidbrücken stellen eines der wichtigsten Elemente der korrekten Proteinfaltung dar und werden in Eukaryonten vorwiegend im oxidierenden Milieu des ER in das naszierende Protein eingeführt. Aus diesem Grunde wurden Proteindisulfid-isomerasen (PDIs) und ER-oxidoreduktin (Ero)-ähnliche Proteine, die die Schlüssel-komponenten der Bildung von Disulfidbrücken in Eukaryonten darstellen, näher untersucht. In S. pombe finden sich insgesamt drei PDI-Homologe (SpPdi1p, SpPdi2p und SpPdi3p) sowie zwei Ero-Homologe (SpEro1a p und SpEro1b p). Mit Ausnahme des nicht glycosylierten SpPdi2p, sind alle Proteine Membran-assoziierte glycosylierte Komponenten des ER. SpPdi2p und SpPdi3p sowie SpEro1a p und SpEro1b p liegen in vivo teilweise in oxidiertem Zustand vor. Des Weiteren konnte gezeigt werden, dass SpEro1b p, nicht jedoch SpEro1a p in der Lage ist, die temperatursensitive S. cerevisiae ero1-1-Mutante funktionell zu komplementieren. Interessanterweise ergab die Untersuchung konservierter Cysteine mittels gerichteter Mutagenese einerseits Unterschiede zwischen SpEro1a p und SpEro1b p sowie andererseits zwischen den S. pombe Ero-Proteinen und den Ero-Proteinen anderer Spezies. Im Gegensatz zu Ero1b p wird Ero1a p durch reduzierenden Stress und Hitzestress induziert. Dies deutet darauf hin, dass SpEro1b p für die Bildung von Disulfidbrücken unter normalen Wachstumsbedingungen nötig ist, während SpEro1a p vornehmlich bei der Adaption der Zellen an Stressbedingungen erforderlich ist. Abschließend konnte gezeigt werden, dass die gesteigerte Expression von SpEro1a p und SpEro1b p zu einer deutlich erhöhten Ausbeute des disulfidhaltigen heterologen Proteins Orf19p-HA führt. Dieser Befund impliziert, dass in S. pombe die Oxidation der Disulfidbrücken für die Faltung von Proteinen vermutlich limitierend ist.

Chapter 1gives a general introduction to the CXXC motif found in natural proteins. It then reviews the studies where disulphides were engineered in various proteins. The various strategies developed to engineer metal binding activity and redox activity are described. The objectives behind engineering the CXXC motif into a protein, such as imparting it novel metal-binding and redox activities, are discussed next. Alternative strategies which achieve the same objectives are described as well. This chapter then introduces the model proteins used in the course of this thesis: maltose-binding protein (MBP) and E. coli. Thioredoxin (Trx). This chapter also briefly discusses the role of signal peptide in protein export. Chapter 2describes the experimental studies and their results in which we introduced the widely occurring cysteine motif CXXC into the maltose binding protein (one-at-a-time, in five alpha-helices, at the N-termini) to test three hypotheses: 1) Does a disulphide bond form at the N-terminus? 2) Does the protein acquire any oxido-reductase activity? 3) Does it acquire new metal-binding properties? The results confirmed: 1) Each cysteine pair forms a stable intrahelical disulphide bond under non-reducing conditions. 2) The five mutant proteins acquire considerable oxidoreductase activity, tested by the insulin aggregation assay. 3) The mutants acquire novel metal-binding properties for Ni2+, Cd2+, and Zn2+ upon reduction. Further, introducing the CXXC motif neither destabilizes the protein nor affects its global structure. Our results demonstrated that introduction of CXXC motifs can be used to probe alpha-helix start sites and to introduce oxidoreductase and metal binding functionality into proteins. Chapter 3describes further experimentson a few of the metal ion binding mutants discussed in the previous chapter. We explore the effect and usefulness of reducing agents (DTT and TCEP) on the binding of metal salts to the CXXC mutants. We also studied the explore of metal salts on the thermal stability of the mutants and show that metal ions bind to the CXXC motif even when the protein is in the unfolded state. The chapter describes the use of an immobilized metal affinity chromatography (IMAC) based method for the purification of MBP mutants.Yields ranging from 60-85% were obtained for thethree MBP mutants. The cysteines were located at different positions in thesethree MBP mutants (MBP 42-45 Cys, MBP 128-131 Cys, and MBP 359-359 Cys mutants). The yields for wild-type MBP, a single cysteine mutant (MBP S211C), a double cysteine mutant (MBP 230, 30) were all below 15%. Chapter 3 also reports a new crystal structure of the MBP356-359 mutant in ligand bound form:it crystallizes as an intermolecular dimer, bonded by two disulfides formed by the cysteines of the CXXC motif. Chapter 4describes the effects of inserting signal peptide sequences on protein folding and expression. We fused the malE and pelB signal sequences at the N-terminus of the model protein thioredoxin and observed that the wild-type and pelB fusion constructs are soluble when expressed, but the malE construct was targeted to inclusion bodies. Nonetheless, it could be refolded in vitro to yield a monomeric product with a secondary structure identical to the wild-type thioredoxin. This chapter also details the thermodynamic stability, aggregation propensity and activity of the purified recombinant proteins in comparison with the wild-type thioredoxin. The presence of the signal sequences reduces the thermodynamic stability and activity of the recombinants and increases their aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, different signal sequences affect protein stability and aggregation differently. Chapter 5describes three different strategies to label a protein at different sites with cysteine-specific fluorophores using MBP as the model. The first strategy exploits the differential accessibility of residues within MBP in its maltose-bound and maltose-free states. The second strategy involves insertion of a 14-amino-acid loop called V3 from the HIV gp120 protein into MBP; anti-V3 antibodies shield the cysteine residue present inside the inserted loop, while we label another cysteine present outside the loop. In the third strategy, we introduce a third cysteine residue onto the background of the MBP mutant already containing a disulphide bridge at the N-terminus of one of its helices (discussed in Chapter 2). We label the third, free cysteine while the cysteines involved in the disulphide bridge remain protected. We observed successful differential labelling using the first strategy and also observed FRET between the fluorophore labels. Similarly, after trying the second strategy we could individually label all the mutants except one. The third strategy based on the triple-cysteine mutant was not successful because the fluorophore we chose (DBM) did not show site specificity and instead labelled all three cysteines. In addition, the triple-cysteine mutant did not even show disulphide-bridge formation.We showed that indeed the V3 loop inserted in MBP binds anti-V3 antibodies and we could individually label all the mutants expect D41C. The third strategy was not successful because unfortunately in the triple cysteine mutant, the fluorophore we chose (DBM) did not show site specificity and labeled all three cysteines. In addition, the disulfide bridge was not found to be present in the triple cysteine mutant. Chapter 6discusses the synthesis, characterization and binding of various maltolipids, (and their corresponding maltose-free controls) to MBP. The maltolipids were synthesised with varying linker lengths and anchor- & head-groups and then used to prepare liposomes and micelles. Although both liposomal and micellar forms could bind to MBP, only the micelles were screened subsequently for their ability to bind to MBP. The binding was assessed using various techniques such as fluorescence spectroscopy, gel filtration and thermal stability assay. We screened the maltolipids and determined how their anchor group, linker length and charge on the head group influences the binding of MBP to micelles formed by these maltolipids.

Die vorliegende Arbeit beschäftigt sich mit der genetischen Optimierung der Spalthefe S. pombe für die biotechnologische Produktion von Fremdproteinen. Hierbei werden vor allem zwei Aspekte näher untersucht, zum einen die Stabilität des zu produzierenden Proteins und zum anderen die Bildung von Disulfidbrücken. Von anderen Organismen ist bekannt, dass die N-terminale AS im Verbund mit einem Lysinrest ein Protein destabilisieren kann. Das Modellprotein vVEGF besitzt an Position 2 einen Lysinrest (K2) und damit ein Hauptmerkmal eines derartigen Destabilisierungselementes. Falls das Protein dem Ubiquitin-vermittelten Abbau unterliegt, ist es wahrscheinlich, dass K2 eine essenzielle Rolle für die Stabilität dieses Proteins spielt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass K2 in S. cerevisiae destabilisierend wirkt, während es in S. pombe keinen destabilisierenden Effekt hat. Dieses Ergebnis spricht dafür, dass es Unterschiede im Ubiquitin-vermittelten Abbau von Proteinen in diesen beiden Hefen gibt. Der Schwerpunkt dieser Arbeit lag auf der Analyse und Optimierung der Bildung von Disulfidbrücken in S. pombe. Disulfidbrücken stellen eines der wichtigsten Elemente der korrekten Proteinfaltung dar und werden in Eukaryonten vorwiegend im oxidierenden Milieu des ER in das naszierende Protein eingeführt. Aus diesem Grunde wurden Proteindisulfid-isomerasen (PDIs) und ER-oxidoreduktin (Ero)-ähnliche Proteine, die die Schlüssel-komponenten der Bildung von Disulfidbrücken in Eukaryonten darstellen, näher untersucht. In S. pombe finden sich insgesamt drei PDI-Homologe (SpPdi1p, SpPdi2p und SpPdi3p) sowie zwei Ero-Homologe (SpEro1a p und SpEro1b p). Mit Ausnahme des nicht glycosylierten SpPdi2p, sind alle Proteine Membran-assoziierte glycosylierte Komponenten des ER. SpPdi2p und SpPdi3p sowie SpEro1a p und SpEro1b p liegen in vivo teilweise in oxidiertem Zustand vor. Des Weiteren konnte gezeigt werden, dass SpEro1b p, nicht jedoch SpEro1a p in der Lage ist, die temperatursensitive S. cerevisiae ero1-1-Mutante funktionell zu komplementieren. Interessanterweise ergab die Untersuchung konservierter Cysteine mittels gerichteter Mutagenese einerseits Unterschiede zwischen SpEro1a p und SpEro1b p sowie andererseits zwischen den S. pombe Ero-Proteinen und den Ero-Proteinen anderer Spezies. Im Gegensatz zu Ero1b p wird Ero1a p durch reduzierenden Stress und Hitzestress induziert. Dies deutet darauf hin, dass SpEro1b p für die Bildung von Disulfidbrücken unter normalen Wachstumsbedingungen nötig ist, während SpEro1a p vornehmlich bei der Adaption der Zellen an Stressbedingungen erforderlich ist. Abschließend konnte gezeigt werden, dass die gesteigerte Expression von SpEro1a p und SpEro1b p zu einer deutlich erhöhten Ausbeute des disulfidhaltigen heterologen Proteins Orf19p-HA führt. Dieser Befund impliziert, dass in S. pombe die Oxidation der Disulfidbrücken für die Faltung von Proteinen vermutlich limitierend ist.