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.

Contexte. Les protéines transmembranaires ont une importance considérable tant au niveau de la survie d'une cellule qu'au niveau de ces interactions avec les autres cellules. En raison de contraintes techniques, la cristallisation de ce type de protéine demeure très complexe, ce qui limite grandement l’exploration de leur structure. Pour contourner ces difficultés, différents outils de prédiction ont été développés,en se fondant originellement sur l'hydrophobicité des régions enfouies dans la membrane. Méthode. L'outil développé repose sur une dérivation de la moyenne d'hydrophobicité calculée sur deux ensembles de taille de fenêtres. Le premier ensemble (G1) contient des petites tailles de fenêtres ce qui correspond à des événements locaux, tandis que le second (G2) correspond à des tailles de fenêtres plus larges, adaptées à la taille des hélices formant certaines protéines transmembranaires. La variation d'hydrophobicité est obtenue en dérivant les moyennes d'hydrophobicité. Un consensus est établi pour chaque groupe, et les résultats sont comparés à un ensemble de protéines transmembranaires cristallisées. Résultats. Les variations d'hydrophobicité G2 sont liées aux extrémités des hélices transmembranaires,tandis que les variations G1 sont en relation avec la limites des structures et certaines irrégularités structurelles.Ces résultats nous ont amené à introduire une nouvelle notion : les unités transmembranaires(TMU). Les TMU consistent en un ensemble de sous-structures qui composent les structures transmembranaires. / Background. Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction oftheir transmembrane (TM) structure by bioinformatics tools provides interesting insights on the topology of these proteins.Method. We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses) in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integralmembrane proteins we defined two levels of analysis : G1-HPulses for sliding windows of n=2 to 6 andG2-HPulses for sliding windows of n=12 to 16.Results. The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new conceptof transmembrane unit (TMU) that groups together transmembrane helices and segments with potentialadjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that correspondedto kinks, partial helices or unannotated structural events. These irregularities could represent key dynamicelements that are alternatively activated depending on the channel status as illustrated by the crystalstructures of the lactose permease in different conformations. Our results open a new way in the understanding of transmembrane secondary structures : hydrophobicity through hydrophobic pulses stronglyimpacts on such embedded structures and is not confined to define the transmembrane status of aminoacids.

Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening. This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively. These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane. A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease. Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria. / <p>At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper nr. 1: Manuscript; Paper nr. 4: Manuscript</p>

Abstract The surface of multicellular organisms is covered with epithelial cells that provide a barrier to the external environment. As part of this barrier function, most epithelia produce apical extracellular matrices (aECMs). The generation of such chemical and physical barriers requires specialized deposition of macromolecules and is likely to involve a spatial and temporal coordination of biochemical activities at the apical surface. A challenging task is thus to characterize key proteins that underlie apical cell surface organization and correct aECM assembly. The Drosophila trachea provides an excellent system to study aECM formation, as they produce an ordered aECM, called the cuticle. The tracheal cuticle is unique by its presence of cuticular ridges, called taenidial folds, which prevent collapse of tracheal tubes while allowing them to expand and contract along their length. A gene called filzig encodes a transmembrane serine protease and is required for taenidial organization. The aim of this research was to further understand Filzig function through characterization of filzig mutants and Filzig protein expression. The results showed that Filzig is expressed in cuticle-producing epithelia as cuticle deposition begins. Moreover, Flz localized to the apical epithelial surface, as well as to the aECM. The apical Flz localization does not reflect the pattern of cuticle ridges, indicating that Flz-localization is not a determinant for taneidial patterning. Instead, Flz might act on extracellular targets that localize to the future taneidial folds. Alternatively, Filzig is involved in a cascade of self-organizing activity of cuticular components to form the regular taenidial folds.

Traditional algorithms for hidden Markov model decoding seek to maximize either the probability of a state path or the number of positions of a sequence assigned to the correct state. These algorithms provide only a single answer and in practice do not produce good results. The most mathematically sound of these algorithms is the Viterbi algorithm, which returns the state path that has the highest probability of generating a given sequence. Here, we explore an extension to this algorithm that allows us to find the k paths of highest probabilities. The naive implementation of k best Viterbi paths is highly space-inefficient, so we adapt recent work on the Viterbi algorithm for a single path to this domain. Out algorithm uses much less memory than the naive approach. We then investigate the usefulness of the k best Viterbi paths on the example of transmembrane protein topology prediction. For membrane proteins, even simple path combination algorithms give good explanations, and if we look at the paths we are combining, we can give a sense of confidence in the explanation as well. For proteins with two topologies, the k best paths can give insight into both correct explanations of a sequence, a feature lacking from traditional algorithms in this domain.

Traditional algorithms for hidden Markov model decoding seek to maximize either the probability of a state path or the number of positions of a sequence assigned to the correct state. These algorithms provide only a single answer and in practice do not produce good results. The most mathematically sound of these algorithms is the Viterbi algorithm, which returns the state path that has the highest probability of generating a given sequence. Here, we explore an extension to this algorithm that allows us to find the k paths of highest probabilities. The naive implementation of k best Viterbi paths is highly space-inefficient, so we adapt recent work on the Viterbi algorithm for a single path to this domain. Out algorithm uses much less memory than the naive approach. We then investigate the usefulness of the k best Viterbi paths on the example of transmembrane protein topology prediction. For membrane proteins, even simple path combination algorithms give good explanations, and if we look at the paths we are combining, we can give a sense of confidence in the explanation as well. For proteins with two topologies, the k best paths can give insight into both correct explanations of a sequence, a feature lacking from traditional algorithms in this domain.

The key control step in E. coli chemotaxis is regulation of CheA kinase activity by a set of four transmembrane chemoreceptors. The receptor dimers can form trimeric complexes (trimers of dimers), and these trimers can be joined by a bridge thought to consist of a CheW monomer, a CheA dimer, and a second CheW monomer. It has been proposed that trimers of receptor dimers may be joined by CheW-CheA dimer-CheW links to form an extended hexagonal lattice that may be the structural basis of the chemoreceptor patches seen in E. coli. The receptor/CheA/CheW ternary complex is a membrane-spanning allosteric enzyme whose activity is regulated by protein interactions. The study presented in this dissertation investigated intermolecular and intramolecular interactions that affect the chemotactic signal processing. I have examined functional interactions between the serine receptor Tsr and the aspartate receptor Tar using a receptor coupled in vitro phosphorylation assay. The results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis. A mutational analysis of the extreme C-terminus (last fifty residues) of Tar is also presented. The results implicate the receptor C-terminus in maintenance of baseline receptor activity and in attractant-induced transmembrane signaling. They also suggest how adaptive methylation might counteract the effects of attractant binding.

This dissertation covers a broad range of research topics all towards the ultimate goal establishing of a novel type of biosensor in which the biocompatible membrane structure reconstituted with functional transmembrane proteins is utilized as the sensing element. It focuses on 1) examining the activity of a model transmembrane protein, bovine rhodopsin (Rho) when reconstituted into stabilized lipid membranes and 2) the instrumentation of a novel type of optical spectroscopy, plasmon waveguide resonance (PWR), which is a surface sensitive technique and its application in sensing biological events.Lipid membrane play crucial roles in cell function. Their biophysical properties affect the activity of a large amount of transmembrane receptors. They are great candidates for biosensing/ biomedical coating. However, the intrinsic instability of natural or fluid membranes prevents them to be used in a device. Studies have been done to show indirect evidence that the activity of Rho maybe maintained in polymerized membrane composed of bis-SorbPC lipids. The activity of Rho reconstituted into vesicular membranes comprised of various lipids was studied by a more direct technique, UV-Vis. It was found Rho activity was maintained to 66% of that in natural Egg PC lipid in the mixture of Egg PC:(poly)bis-SorbPC (1:1 mol:mol) as opposed to minimal values in 100 % (poly)lipids.A new type of spectral PWR was developed. The working concept, technical characterization and comparisons with similar techniques were discussed and compared in this work. A modified version of angular PWR in which lipid bilayers were formed by vesicle fusion was also developed. This method excludes possible effects from a high boiling point organic solvent on either the lipid bilayer itself or the membrane proteins associated with it. A calculating program NphaseAll for PWR was developed to do predictions of waveguide properties can be made to provide guidance for waveguide design. Theoretical calculations were done for PWR and experimental results were compared with the theoretical predictions.PWR was used to detect the formation of a biological lipid membrane, the association of alpha synuclein with membranes and the binding activity of human melanarcortin to its ligands in fluid and polymerized/dried membranes.

This thesis describes the expression of three human seven transmembrane receptors: the M2 Muscarinic; H1 Histamine and 5HT2A Serotonin receptors, in the baculovirus/insect cell expression system. Purification trials werre conducted on the M2 Muscarinic and H1 Histamine receptors. Preliminary crystallisation attempts were made with the H1 receptor.

Members of the membrane-spanning four-domains, subfamily A (MS4A) family are small polypeptides that share the structural features of four-transmembrane domains and unevenly sized extracellular loops. The family includes CD20, FcεRIβ and HtM4, plus a number of relatively uncharacterised proteins / predicted proteins. MS4A proteins are discussed in relation to other protein families, such as the tetraspanins, that are also characterised by four-transmembrane domains. The aim of this study was to identify the cell and tissue distribution, subcellular localisation, and function of a newly discovered member of the MS4A family, hepatocellular carcinoma-associated antigen 112 (HCA112). At a subcellular level, HCA112 was found on the plasma membrane of transfected COS-7 cells, and also within the Golgi complex, trans-Golgi-network, and early endosomes. The molecule is orientated such that the large loop is extracellular and the Nand C-terminal domains are cytoplasmic. The presence of HCA112 associated with components of the endocytic pathway raised the question of whether some originated from the surface membrane. Antibody was used to label a HA epitope tag engineered into the large extracellular loop of HCA112, and the bound antibody was tracked through early endosomes to the recycling compartment. Here it co-localised with internalised transferrin, indicating strongly that HCA112 is internalised via clathrin-dependent mechanisms. Several endocytic sorting motifs within the intracellular domains of HCA112 were investigated for their ability to direct internalisation of HCA112. Deletion of a di-leucine motif was found to slow but not prevent endocytosis, suggesting that it is involved in endocytosis of HCA112, although not essential for the process. When HCA112 expression constructs featuring N- and C-terminal domain truncations were examined, it was found that the N-terminal tail does not affect the subcellular localisation or trafficking of HCA112, while deletion of the C-terminal intracellular domain resulted in retention of the mutant protein in the ER. HCA112 has a wide tissue distribution and is highly expressed in the lining/covering and parenchymal epithelium of some tissues, proximal renal tubules, ductal epithelium in a number of organs, endothelial cells, some steroidogenic endocrine cells, adipocytes, smooth muscle cells, follicular dendritic cells and macrophages. The expression of HCA112 by a wide range of cell types suggests that its function(s) has general importance and is not limited to any specific cell type(s). After reflection on the functions of the HCA112-expressing cells, a common theme that emerged was one of endocytic activity. This lead to speculate that one function of HCA112 might be related to uptake of macromolecules, for instance, in antigen processing and presentation. This might be a general function, such as facilitating uptake of other cell membrane proteins, or directing the traffic of endocytic vesicles. It was noted that HCA112 has a similar cell and tissue distribution to the scavenger receptor and fatty acid translocase FAT/CD36 (Zhang et al., 2003). Furthermore, in cells co-transfected with HCA112 and FAT/CD36, the two molecules co-localise in early endosomes and co-immunoprecipitate, suggesting that the molecules physically and spatially associate. Thus, HCA112 could be involved with (or complement) FAT/CD36 in its functions as a long chain fatty acid transporter and scavenger receptor. A proteomics study of proteins that co-immunoprecipitated with HCA112 detected putative interactions with a number of proteins. These included LR8, transferrin receptor, interferon induced transmembrane proteins 2 and 3, Calpain-6, stomatin, PDGF α receptor, and heat shock 70 kDa protein 8 (HSPA8, formerly known as clathrin un-coating ATPase). Of these, LR8 and the transferrin receptor were investigated in more detail. The results provide strong evidence that HCA112 forms a novel complex with LR8, and that this may be involved in macromolecule internalisation or trafficking of membrane proteins, such as FAT/CD36 or the transferrin receptor. In the case of the transferrin receptor, this traffic appears to involve the clathrin-dependent pathway, but it is possible that when HCA112 is associated with FAT/CD36, it functions within lipid raft domains. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375454 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009