One approach to protein structure prediction is to first predict from sequence, a thresholded and binary 2D representation of a protein's topology known as a contact map. Then, the predicted contact map can be used as distance constraints to construct a 3D structure. We focus on the latter half of the process and aim to obtain a set of non-binary distance constraints from contacts maps. This thesis proposes an approach to extend the traditional binary definition of “in contact” by incorporating fuzzy logic to construct fuzzy contact maps from a set of contact maps at different thresholds, providing a vehicle for error handling. Then, a novel template-based similarity search and distance geometry methods were applied to predict distance constraints in the form of a distance map. The three-dimensional coordinates were then calculated from the predicted distance constraints. Experiments were conducted to test our approach for various levels of noise. As well, we compare the performance of fuzzy contact maps to binary contact maps in the framework of our methodology. Our results showed that fuzzy contact map similarity was indicative of distance map similarity. Thus, we were able to retrieved similar distance map regions using fuzzy contact map similarity. The retrieved distance map regions provided a good starting point for adaptation and allowed for the extrapolation of missing distance values. We were thus able to predict distance maps from which, the three-dimensional coordinates were able to be calculated. Testing of this framework on binary contact maps revealed that fuzzy contact maps had better performance with or without noise due to a stronger correlation between fuzzy contact map similarity and distance map similarity. Thus, the methodology described in this thesis is able to predict good distance maps from fuzzy contact maps in the presence of noise and the resulting coordinates were highly correlated to the performance of the predicted distance maps. / Thesis (Ph.D, Computing) -- Queen's University, 2012-05-23 13:59:28.12

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The activation of the protein tyrosine kinase (PTK) ZAP-70 is fundamental to T cell receptor (TCR) signal transduction. TCR engagement induces raft-association of ZAP-70 and juxtaposes the cytoplasmic ZAP-70 with the raft-enriched Lck, which phosphorylates and activates ZAP-70. The active ZAP-70, cooperatively with Lck, initiates multiple intracellular pathways eventually leading to T cell activation and IL-2 production. Here, we describe the serine phosphorylation on ZAP-70 on the highly conserved S520DVWS524 motif, and investigate its role in coupling ZAP-70 with TCR signal transduction.

In normal cells, the p53 tumour suppressor induces cell cycle arrest or apoptosis in response to a variety of stresses, including DNA damage and ectopic oncogene expression. However, cellular pathways controlled by p53 are compromised in virtually all cancers. Defining the mechanisms regulating p53 activity in normal and tumour cells has therefore been a major priority in cell biology and cancer research. / In this study, we characterized two important regulatory mechanims of p53 activity: (i) Human papillomavirus (HPV) E6 interaction and (ii) alternative splicing. Recognized as the major etiological agents for cervical cancer, the oncogenic potential of HPVs correlates with their ability to target p53 for degradation. This study demonstrates that both p53 and HPV-18 E6 are exported from the nucleus when co-expressed, via a process that involves the C-terminal nuclear export signal (NES) of p53. However, neither nuclear export nor the p53 C-terminal NES is required for HPV-18 E6-mediated ubiquitination or degradation of p53. / This study also demonstrates that both low- and high-risk HPV E6 proteins are degraded by the ubiquitin-proteasome pathway, and thus provides an explanation for the low levels of E6 detected in cervical cancer cells. / Also reported in this study is a novel mechanism of p53 regulation arising through alternative splicing. This novel mRNA encodes a N-terminal deleted isoform of p53, termed p47. As demonstrated within, p47 does not supress cell viability but impairs both p53-mediated transcriptional activity and growth suppression. Interestingly, p47 increases both p53 monoubiquitination and nuclear export. We propose that p47 induces nuclear export of p53 by a mechanism involving monoubiquitination, as supported by recent findings from Li and colleagues (2003). The p47 protein also protects p53 from both Mdm2- and HPV-18 E6-mediated degradation. A number of cancers display abnormal localization of wildtype p53, and it will be important to examine the role of p47 in these tumours. / Taken together, the regulation of p53 activity by both HPV E6 and the alternative splice variant p47 involves alterations in p53 ubiquitination status, protein stability, and cell localization. Insight gained into these negative regulatory mechanisms may aid in the design of therapeutic strategies for reactivating wild-type p53 in HPV-associated and non-associated cancers.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

The Major Intrinsic Proteins are represented in all forms of life; plants, animals, bacteria and recently archaebacteria have all been shown to express at least one member of this superfamily of integral membrane proteins. We have overexpressed the E. coli aquaglyceroporin, glycerol facilitator (GlpF), to use as a model for studying membrane protein structure, folding and stability. Understanding membrane protein folding, stability, and dynamics is required for a molecular explanation of membrane protein function and for the development of interventions for the hundreds of membrane protein folding diseases. X-ray analysis of GlpF crystals shows that the protein exits as a tetramer in the crystallized state [1]. However, preparations of stable aqueous detergent solutions of GlpF in its native oligomeric state have been difficult to make; the protein readily unfolds and forms non-specific aggregates in many detergents. Here, I report the study of the structure and stability of the glycerol facilitator in several detergent solutions by blue native and sodium dodecyl sulphate polyacrylamide gel electrophoresis, circular dichroism, and fluorescence. For the first time, stable protein tetramers were prepared in two different detergent solutions (dodecyl maltoside (DDM) and lyso-myristoyl phosphatidylcholine (LMPC)) at neutral pH. Thermal unfolding experiments show that the protein is slightly more stable in LMPC than in DDM and that the thermal stability of the helical core at 95oC is slightly greater in the former detergent. In addition, tertiary structure unfolds before quaternary and secondary structures in LMPC whereas unfolding is more cooperative in DDM. The high stability of the protein is also evident from the unfolding half-life of 8 days in 8 M urea suggesting that hydrophobic interactions contribute to the stability. The GlpF tetramers are less resistant to acidic conditions; LMPC-solubilized GlpF shows loss of tertiary and quaternary structure by pH 6, while in DDM the tertiary structure is lost by pH 5, however the tetramer remains mostly intact at pH 4. The implications of thermal and chemical stress on the stability of the detergent-solubilized protein and its in vivo folding are discussed.

Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.