The P22 c2 repressor protein (P22R) binds to DNA sequence-specifically and helps direct the temperate lambdoid bacteriophage P22 to the lysogenic developmental pathway. To gain insight into its DNA binding mechanism, we solved the 1.6 Å x-ray structure of the N-terminal domain (NTD) of P22R in a complex with a DNA fragment containing the synthetic operator sequence [d(ATTTAAGATATCTTAAAT)]2 This operator has an A-T at position 9L and T-A at position 9R and is termed DNA9T. Van der Waals interactions between protein and DNA appear to confer sequence-specificity. The structure of the P22R NTD – NA9T complex suggests that sequence-specificity arises substantially from interaction of a valine with a complementary binding cleft on the major groove surface of DNA9T. The cleft is formed by four methyl groups on sequential base pairs of 5' TTAA 3'. The valine cleft is intrinsic to the DNA sequence and does not arise from protein-induced DNA conformational change. Protein-DNA hydrogen bonding plays a secondary role in specificity.

One of the pressing needs in the field of disease biomarker discovery is new technologies that could allow high performance protein analysis in different types of clinical material, such as blood and solid tissues. This thesis includes four approaches that address important limitations of current technologies, thus enabling highly sensitive, specific and parallel protein measurements. Paper I describes a method for sensitive singleplex protein detection in complex biological samples, namely solid phase proximity ligation assay (SP-PLA). SP-PLA exhibited improved sensitivity compared to conventional sandwich immunoassays. We applied SP-PLA to validate the potential of GDF-15 as a biomarker for cardiovascular disease.   Paper II describes ProteinSeq, a multiplexed immunoassay based on the principle of SP-PLA, for parallel detection of 36 proteins using next-generation sequencing as readout. ProteinSeq exhibited improved sensitivity compared to multiplexed sandwich immunoassays, and the potential to achieve even higher levels of multiplexing while preserving a high sensitivity and specificity. We applied ProteinSeq to analyze 36 proteins, including one internal control, in 5 μl of plasma samples in a cohort of patients with cardiovascular disease and healthy controls. Paper III describes PLA-DTM, a strategy for recording all possible interactions between sets of proteins in clinical samples. Individual proteins and their interactions are first encoded to dual barcoded DNA by PLA, and the barcodes are interrogated by a method named dual tag microarray (DTM). We applied the method for studying interactions among protein members of the NFκB signaling pathway. Paper IV describes a novel probing strategy for analyzing individual biomolecules in solution or in situ. The technique employs a new class of probes for unfolding proximity ligation assays - uPLA probes. The probes are designed so that each probe set is sufficient in forming and replicating circular DNA reporter, without interactions among themselves when incubated with the sample. The uPLA probing strategy provides ease in the design of multiple probe sets in parallelized assays while enhancing the specificity of detection. We used the uPLA probes to detect various targets, including synthetic DNA and cancer-related transcripts in situ.

Bibliography: p. 96-108. / 108, [31] p., [9] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The major aim of this thesis is the identification and characterisation of Polycomblike (PCL) protein interactors. The study analyses the ability of PCL to bind directly to DNA anchoring the Pc-G complex to the genes which they repress. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1997

The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins – syntaxin4, SNAP23 and VAMP2 – and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.

[Truncated abstract] Neuregulins (NRG’s) are pleiotropic growth factors that participate in a wide range of biological processes. The family of membrane-bound growth factors bind to and activate ErbB receptors on adjacent target cells, mediating multiple biological processes. NRG-1, NRG-2 and NRG-3 are all highly expressed in the nervous system, where it has been shown that NRG-1 is important for neuronal development, migration, synapse formation and glial cell proliferation. Little is known, however, on the specific roles of NRG-2 and NRG-3, although it is apparent that despite similar expression patterns and overlapping receptor specificity, NRG-2 and NRG-3 do not compensate for the loss of NRG-1 and mediate their own distinct activities. … Subcellular localisation experiments showed that this domain is important for trafficking of the fulllength protein to various intracellular compartments in an activity dependent manner. In addition, the ICD is required to elicit a cell death response in cultured cells and provoke an elevated α-amino-3-hydroxyl-5-methylisoxazole-4-propionate (AMPA) response in organotypic neuronal cultures following transient expression of NRG-3. A yeast two-hybrid screen identified 14-3-3ζ and PICK1 as two proteins that interacte with the human NRG-3 ICD. These interactions were confirmed both in vitro and in vivo, and were further characterised at a molecular level. This study demonstrates the ability of NRG-3 to mediate signal transduction through a biologically active ICD; a conclusion supported by identifying cytoplasmic proteins that interact with the ICD. These observations point to an additional layer of complexity where bi-directional signalling contributes to the full repertoire of NRG-3 functions.

[Truncated abstract] Hereditary Motor and Sensory Neuropathy Lom (HMSNL) is a severe autosomal recessive peripheral neuropathy, the most common form of demyelinating Charcot-Marie-Tooth (CMT) disease in the Roma (Gypsy) population. The mutated gene, N-myc downstream-regulated gene 1 (NDRG1) on chromosome 8q24, is widely expressed and has been implicated in a wide range of processes and pathways. In this study we have aimed to assess the overall contribution of this gene to the pathogenesis of peripheral neuropathies, in cases where the most common causes of CMT disease havebeen excluded, as well as to gain clues about its function through the identification of its interactions with other proteins. Sequence analysis of NDRG1 in 104 patients with CMT disease and of diverse ethnicity identified one novel disease-causing mutation, IVS8-1G>A (g.2290787G>A), which affects the splice-acceptor site of IVS8 and results in the skipping of exon 9 . . . The results suggest a defect in Schwann cell lipid trafficking as a major pathogenetic mechanism in CMT4D. At the same time, database searches showed that the chromosomal location of NDRG1 coincides with a reported High-Density Lipoprotein-Cholesterol Quantitive Trait Locus (HDL-CQTL) in humans and in mice. A putative role of NDRG1 in the general mechanisms of HDL-mediated cholesterol transport was supported by biochemical studies of blood lipids, which revealed an association between the Gypsy founder mutation, R148X, and decreased HDL-C levels. These findings suggest that while peripheral neuropathy is the drastic result of NDRG1 deficiency, the primary role of the protein may be related to general mechanisms of lipid transport⁄metabolism.

Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 14 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Includes bibliographical references. Also available in print form.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 83-95).