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Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ; cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The recombinant expression of heterologous proteins in microorganisms, such Escherichia coli, is often improved by producing the protein of interest translationally linked to another, often unrelated, protein giving rise to a "fusion protein" construct. For many applications it is desirable or imperative to separate the extraneous material from the protein of interest. An increasingly popular approach to this task is the use of site-specific endoproteases 10 excise the protein product. A number of commercially available site-specific proteases exist, but many are not capable of generating an authentic N-terminus for the product, display unsatisfactory specificity leading to adventitious cleavage of the product, or they are unsuitable for an industrial process. Mutants of the serine protease a-Lytic protease have been shown to satisfy many of the criteria for an industrially suitable protease and have been applied to the cleavage of some important fusion proteins used in the production of members of the Insulin-like Growth Factor (IGF) family Lacking from these examples, however, is any viable proteolytic solution for the liberation of human IGF-I from fusion proteins. This has been primarily attributed to the Proline bearing N-terminal tripeptide sequence of this protein, which is known to be refractory to the activity of many site-specific proteases. It has been suggested that in the generation of two combinatorial mutant libraries of a-Lytic protease, the preference for amino acids C-terminal to the cleavage site may have been altered. It is the purpose of this work to first determine if such an alteration has been made in any of the mutants so as to allow cleavage immediately before the N-terminus of human IGF-1, and then to task the lead mutant(s) to the cleavage of the full-length fusion protein. All members of the two mutant libraries were cultured and their activity confirmed and quantified against a generic B-casein substrate in a high-throughput assay. A second high-throughput technique was then employed to query the mutant proteases for their ability to catalyse proteolysis at the required sequence in a peptide model. Finding that many mutants appeared successful at this task, the findings were verified on a longer peptide model of the cleavage site. Initially the yields achieved by cleavage of the full-length IGF-1 fusion protein by a lead candidate mutant a-Lytic protease were not sufficient to satisfy the requirements of an industrial process, despite alteration of the reaction conditions. However, the insight gained from these reactions could be applied to the redesign of the protein structure around the intended site of cleavage, significantly improving site-specific proteolysis. The IGF-1 generated by this cleavage has been shown to be bioequivalent to commercial reference standard to cultured mammalian cells and the yield of this process is approximately 5-fold improved over the existing cleavage system. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325381 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

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The mannose-binding lectin from snowdrop (Galanthus nivalis agglutinin; GNA) was produced in Escherichia coli and purified as a functional protein after denturation/renaturation. Incorporation of the four extra C-terminal residues recently revealed from X-ray crystallographic data demonstrated that these residues increase binding to the glycoprotein carboxypeptidase Y. However, no differences in activities were observed in haemagglutination assays when compared to native GNA and toxicity towards rice brown planthopper (Nilaparvata lugens', BPH) in artificial diet bioassays was unaltered. Site-directed mutagenesis of the carbohydrate-binding site of GNA provided evidence of a direct correlation between the binding potential of GNA to BPH gut glycoprotein 'receptors' and the toxicity levels of GNA towards BPH nymphs. Functional recombinant plant lectins GNA and PHA (Phaseolus vulgaris agglutinin) were expressed in Pichia pastoris using native signal peptides or the Saccharomyces a-factor prepro-sequence to direct secretion. The a-factor prepro-sequence was inefficiently processed unless Glu-Ala repeats were added at the C-terminal end. In the latter case, removal of the Glu-Ala repeats was itself inefficient leading to recombinant lectins with heterogenous N-termini. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed and fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs in Pichia. A fusion protein containing both GNA and GFP (GNA-GFP) was expressed in Pichia pastoris. Simultaneous dual activities (i.e. carbohydrate binding and fluorescence) of recombinant GNA-GFP were demonstrated. Partial cleavage in the linker region resulted in co-purification of GNA which increased the binding activity of the fusion protein. Selective binding of GNA-GFP to haemocytes in the haemolymph of Lacanobia oleracea was observed, both in vitro and when the protein was fed to insects in diet.

Silk is a natural protein produced by members of the class Arachnida (over 30,000 species of spiders) and by several worms. Silk-based materials have been investigated for medical and biotechnological applications for many years. Although silkworm silk has been studied extensively because of ready availability of the protein, lately the advancements in recombinant technology has made production of spider silk proteins increasingly available. Due to the characteristics like biocompatibility, biodegradability and mechanical strength, silk is highly desirable as a biomaterial for medical purpose. Along with this, techniques for functionalization, has further aided in the development of silk into highly sophisticated material for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein Masp2. Two distinct approaches were used, chemical modification and genetic fusion. In the first modification, we created an infection responsive silk nanospheres by chemically grafting a thrombin sensitive peptide to the silk protein encapsulating antibiotic. These particles were then evaluated for in vitro infection responsive drug release and antimicrobial activity. From these assessments, we found that these particles can release the drug effectively in the presence of infection providing the evidence that these particles are enzyme responsive and can be used to formulate targeted drug release. In the second modification, spider silk was genetically modified with a heparin binding peptide to create a fusion protein which can prevent both thrombosis and infection simultaneously. This fusion protein was evaluated for its heparin binding ability and anticoagulant properties in its solution form. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property. After establishing these properties, next this fusion protein was utilized as a coating for hemodialysis catheter. Deposition of coating was evaluated after which anticoagulant and anti-infective properties of the protein as a coating material was investigated. This thesis provides evidence of successful production of a recombinant silk-based biopolymer that can be chemically and genetically embedded with a various functional motif to create a hybrid product for different applications.

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Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.

Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Studies that replace or mutate the TM domain of the F protein of several viruses implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was used to demonstrate that isolated TM domains of HeV F protein associate in a monomer-trimer equilibrium. To determine factors driving this association, we analyzed the sequence of several paramyxovirus F protein TM domains and found a heptad repeat of β-branched residues. Analysis of the HeV F TM domain specifically revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein expression and pre-fusion conformation. To further understand the role of the TM domain, the TM domain was targeted as a potential modulator of F protein stability and function. Exogenous HeV F TM constructs were co-expressed with the full length F protein in Vero cells to analyze the effects on protein expression. Co-expression of the exogenous HeV F TM constructs dramatically reduced the expression of HeV F. However, the co-expression of exogenous HeV F TM constructs with a different paramyxovirus F protein, PIV5 F, did not strongly affect PIV5 F expression levels, suggesting that the interaction of the exogenous TM constructs is specific. Fusion assays revealed that HeV F TM constructs dramatically reduced HeV F, but not PIV5 F fusion activity. We hypothesize that the short exogenous HeV TM constructs associate with the TM domain from full-length HeV F, resulting in pre-mature triggering or protein misfolding. The work presented here demonstrates that specific elements in the TM domain contribute to TM association and pre-fusion protein stability. Furthermore, targeting these interactions may be a viable approach for antiviral development against this important pathogen.