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Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them. / At the time of the doctoral defense the following publications were not published and had a status as follows: Paper 2: In press; Paper 4 Manuscript.

The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation. / In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein. / Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.

Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.

[Truncated abstract] Infectious and allergic diseases of the respiratory tract are major contributors to global mortality, morbidity and economic burden. Bacterial infections such as pneumonia and otitis media are important diseases, especially in children, while allergic diseases such as asthma and allergic rhinitis afflict up to 30% of the world's population. A confounding aspect of respiratory disease is the evidence of a complex relationship between respiratory allergy and respiratory infection, with infection suggested to both promote and prevent the pathogenesis of allergic disease. Additionally, allergy is a risk factor for bacterial infection such as otitis media, pneumonia and sinusitis, while respiratory infection can exacerbate allergic symptoms. Given the burden of bacterial respiratory disease and respiratory allergy, the development of preventative treatments for these diseases is needed and will benefit from clearer knowledge of the underlying immune mechanisms. This thesis aimed to to extend current knowledge by using Pasteurella pneumotropica, a similar bacteria to the human pathogen nontypeable Haemophilus influenzae (NTHi), to study respiratory infection and protective anti-outer membrane protein (OMP) immunity as well as the interaction of respiratory infection and allergic inflammation. Homologues of the important NTHi vaccine candidates P4, P6, P26 and D15 were found to be encoded by P. pneumotropica and a high level of amino acid sequence identity was noted between the different P. pneumotropica strains, as well as between other Pasteurellaceae members. ... In contrast, anti-P6his serum antibodies transferred to naïve mice did not confer protection. These results suggested that T-cell–mediated mechanisms were involved in P6his-mediated protection, and showed that the P. pneumotropcia model was useful for elucidating protective mechansims. The interaction of P. pneumotropica infection and papain-induced allergy was studied to investigate immune mechanisms underlying respiratory infection and allergy. Mice with ongoing allergic inflammation were intranasally challenged with bacteria and exhibited reduced pulmonary bacterial numbers, prolonged eosinophilia in the lungs and the induction of Th2 cytokines in the BALF, compared to nonallergic, infected mice. This suggested a protective role for allergic inflammation in this model. The effect of papaininduced inflammation on mice colonised by P. pneumotropica was also examined and allergic inflammation appeared to worsen infection in colonised mice. This suggested that allergic inflammation may also have a role in promoting infection in this model. In conclusion, this thesis explored mechanisms involved in vaccine-mediated immunity and the interaction of respiratory infection and allergy using a P. pneumotropica infection in its natural host. It was shown that intranasally administered recombinant P6 and P4 protected mice from lung infection, which justifies the inclusion of these OMPs as NTHi vaccine candidates. Additionally, it was demonstrated that the interaction of allergy and respiratory infection modulated immune responses. Overall, these results emphasize that a clearer understanding of the complex mechanisms underlying these interactions is required, and may be aided by the development of suitable animal models.

Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/07/24). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [100]-113).

Thesis (M.S.)--University of North Carolina Wilmington, 2008. / Title from PDF title page (viewed September 24, 2008) Includes bibliographical references (p. 58-59)

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 117-126). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;