The extracellular matrix (ECM) is a complex of proteins and carbohydrates that not only provides a structural support between tissues but also functions in a wide variety of cellular activities. ECM proteins are large, complex proteins with many repeating domains. Individual domains can be analyzed for the investigation of structure-function relationships. This thesis focuses on two ECM proteins of interest: N-terminal agrin (NtA) and the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Agrin is an important ECM protein involved in postsynaptic differentiation at the neuromuscular junction, mediated by binding in the NtA domain. In agrin, the NtA domain is followed by nine follistatin-like (FS) domains. Structural studies showed a novel interdomain disulfide bridge between the NtA and first FS domain in agrin. This disulfide bridge compensates for a seven residue splice insert in the C-terminus of NtA, suggesting that the interdomain disulfide bond may be necessary for the proper folding of agrin. COMP is another important ECM protein that is found in cartilage, tendon, and ligament. It is a homopentamer held together by disulfide bonds in the central coiled-coil oligomerization domain. Previous structural studies demonstrated that COMPcc forms a pentameric α-helical coiled-coil structure containing a 73Å-long cavity with a diameter of 2–6Å that is capable of binding hydrophobic compounds. This binding capacity of COMPcc was investigated and the high-resolution crystal structures of COMPcc in complex with five naturally-occurring fatty acids were solved. Additionally, the binding properties of COMPcc in solution were investigated through the use of fluorescence spectroscopy. Both the x-ray crystallographic and solution data reveal that binding favourability of fatty acids to COMPcc is driven by length of the methylene tail and degree of unsaturation. These results suggest the possibility of COMPcc to be used in targeted drug delivery systems.

The interaction of deoxyribonucleic acid (DNA) and cellular proteins is absolutely central to any biological understanding of DNA replication, transcription, and even gene regulation. Because an incumbent protein latches not onto the four bases but onto the backbone phosphate groups of the nucleic acid, backbone dynamics directly pertain to an understanding of basic cell processes. Studies have unambiguously proven that DNA exists in a balance of two conformations, BI and BII, defined by the difference in their backbone torsion angles. A given DNA sequence expresses a preference for either BI or BII, though both exist in most samples (and are presented as a ratio). Factors affecting that ratio include flanking sequence and methylation. When a DNA sample is methylated, which occurs at a cytosine, backbone dynamics at that site and perhaps even its neighbors are theoretically quenched due to the steric strain of a large attached group. DNA methylation is implicated in cancer diagnosis by new studies focusing on hypermethylation in CpG islands, This thesis uses solid-state deuterium NMR to study the backbone dynamics of the Dickerson dodecamer, [d(CGCGAATTCGCG)]2, which was the first synthetic BII conformer successfully crystallized (allowing for analysis in the solid state) and which contains the EcoRI binding site, GAATTC. This molecule is a good model system because a massive amount of information has been gathered on it not only using NMR, both high-resolution and solid-state, but also using x-ray diffraction, electron paramagnetic resonance, and all-atom molecular dynamics simulation. This thesis research shows the quenching of backbone dynamics due to C9 methylation.

The extracellular matrix (ECM) is a complex of proteins and carbohydrates that not only provides a structural support between tissues but also functions in a wide variety of cellular activities. ECM proteins are large, complex proteins with many repeating domains. Individual domains can be analyzed for the investigation of structure-function relationships. This thesis focuses on two ECM proteins of interest: N-terminal agrin (NtA) and the coiled-coil domain of cartilage oligomeric matrix protein (COMPcc). Agrin is an important ECM protein involved in postsynaptic differentiation at the neuromuscular junction, mediated by binding in the NtA domain. In agrin, the NtA domain is followed by nine follistatin-like (FS) domains. Structural studies showed a novel interdomain disulfide bridge between the NtA and first FS domain in agrin. This disulfide bridge compensates for a seven residue splice insert in the C-terminus of NtA, suggesting that the interdomain disulfide bond may be necessary for the proper folding of agrin. COMP is another important ECM protein that is found in cartilage, tendon, and ligament. It is a homopentamer held together by disulfide bonds in the central coiled-coil oligomerization domain. Previous structural studies demonstrated that COMPcc forms a pentameric α-helical coiled-coil structure containing a 73Å-long cavity with a diameter of 2–6Å that is capable of binding hydrophobic compounds. This binding capacity of COMPcc was investigated and the high-resolution crystal structures of COMPcc in complex with five naturally-occurring fatty acids were solved. Additionally, the binding properties of COMPcc in solution were investigated through the use of fluorescence spectroscopy. Both the x-ray crystallographic and solution data reveal that binding favourability of fatty acids to COMPcc is driven by length of the methylene tail and degree of unsaturation. These results suggest the possibility of COMPcc to be used in targeted drug delivery systems.

<p>Bacteria recycle pre-existing peptidoglycan in order to minimize the de novo synthesis of peptidoglycan precursors. The recycling pathway is under study for its chemotherapeutic target potential. Anhydromuramic acid kinase (AnmK) is part of this recycling pathway and catalyzes the dual hydrolysis/phosphorylation of anhMurNAc to MurNAc-6-P. This enzyme has been discovered and introduced, but only minimally characterized. Therefore, the overarching goal of this work was to clone, express and purify AnmK to homogeneity; perform further kinetic characterization; solve the open, closed and transition state mimic-bound conformations of AnmK by x-ray crystallography; and develop a putative mechanism based on the accumulated research findings and <super>18</super>O-labeling studies.</p><p>The anmK gene was successfully cloned as a hexahistidine fusion protein and overexpression was optimized. After exhaustive trials, a final purification scheme was designed to yield homogeneous AnmK in three chromatographic steps and in less than 36 hours. Additionally, the synthesis of both anhMurNAc and a pseudosubstrate (anhGlcNAc) were carried out in 35% and 77% overall yield, respectively. The synthesis of these compounds allowed for both kinetic characterization and structural studies. </p><p>To this end, the structure of de novo AnmK was solved using SAD and high-resolution (1.9 &Aring;) data. Also, an ATP analog (ANP) and anhMurNAc substrate-bound, closed conformation structure (1.95 &Aring;) was solved. These structures elucidated an 11&deg; domain closure of the enzyme upon substrate binding and also revealed the active site geometry to be used to determine potential molecular determinants of specificity. </p><p>Insights into the kinetic mechanism of AnmK were then gathered using multiple techniques. First, the structure of AnmK (2.5 &Aring;) was solved the with a known transition state analog, the MgADP-vanadate complex. Following this structure, which sheds light on the potential importance of a residue other than the catalytic base (Asp187), isotopic labeling was performed with H₂<super>18</super>O. Using NMR and MS, the regiochemical selectivity of AnmK hydrolysis to impart the solvent derived oxygen at C1 was established. Additionally, this was carried out with stereochemical preference to create the &alpha;-anomer of the carbohydrate product. This regiochemistry and stereospecificity drove the design of our putative concomitant hydrolysis/phosphorylation mechanism but we are not able to rule out the formation of a transient phosphoenzyme intermediate.</p><p>This research can be applied to the immediate goal of understanding the function of a single, novel enzyme with unique chemistry and the clarification of the AnmK mechanism will facilitate future investigation into enzymes with dual hydrolase/kinase functionality. Furthermore, this research contributes to understanding of the complex bacterial peptidoglycan layer in order to harness new ideas for developing antibiotics.</p> / Dissertation

Lipopolysaccharide (LPS), one of the main components of Gram-negative bacterial cell walls, is a potent endotoxin. Structures of the unique protective monoclonal antibody (mAb) WN1 222-5 in complex with Escherichia coli R2 and R4 LPS core regions show that recognition occurs in a manner similar to the innate immune receptor Toll-like receptor 4 (TLR4). Inner core LPS is shown to exist in a conserved epitope with multiple intramolecular interactions that allows the conserved epitope to bind strongly to mAb WN1 222-5. The structure of mAb FDP4, directed against truncated E. coli J-5 LPS, shows a deep pocket combining site specific for a terminal epitope that does not allow room for wild type (wt) LPS. Research into these anti-LPS binding mAbs opens up new avenues for potential septic shock therapy. The explosion of new techniques and bright x-ray sources in the 80’s and 90’s led to rapid advancement of protein x-ray crystallography; however, structure determination on some of the most important problems is now stalled due to the general inability to grow crystals of sufficient size. Recent advances in electron microscopy (EM) technology has led to improved beam characteristics, which has allowed the initiation of research to develop EM as a viable alternative to x-ray crystallography. In this research, method development using standard equipment to explore potential avenues for analysing three-dimensional protein crystals via EM has been explored. / Graduate / 0982 / 0487 / 0537 / kgomery@uvic.ca

The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

Single particle electron cryomicroscopy (cryo-EM) is a rapidly developing structural biology technique for the study of macromolecular protein complexes. Presently, cryo-EM fills an important niche by facilitating acquisition of 3-D structures of protein complexes not amenable to structure determination by other techniques. Expansion of cryo-EM beyond this niche requires continued improvement in the types of specimens that can be studied as well as the final resolutions achieved. Two studies were undertaken to address these issues. The first examined resolution limitations by quantifying the effect of beam-induced motion in images of beam-sensitive paraffin crystals. The second explored the possibility of using cryo-EM to study the interaction of small effector proteins with a large multi-protein complex, V-ATPase. The results of these studies exposed the fact that fundamental aspects of the imaging and specimen preparation processes remain poorly understood and must be addressed to facilitate future improvements in cryo-EM structure determination.

Endotoxin recognition by the innate immune receptor CD14 is a critical part of the innate immune system’s early detection and activation of the inflammatory response during microbial invasion. The differential recognition and high affinity binding of endotoxins from gram-negative and gram-positive bacteria is performed by the innate immune receptor CD14. Upon endotoxin binding, CD14 transfers the specific endotoxins to a Toll-like receptor signaling complex, which is responsible for initiating the intracellular signaling cascade. In the presence of overwhelming infection, the effects of CD14 lead to the over-activation of the inflammatory response, which results in the life threatening condition known as sepsis. Preparation of a 15N isotopically labeled truncated version of soluble CD14, using Pichia pastoris, allowed direct structural observation of the binding interaction between CD14 and two endotoxin ligands, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, respectively using solution NMR spectroscopy. These studies revealed that CD14 uses both a common set of residues, and endotoxin specific subsets of residues, to bind LPS and LTA. To further investigate the structural features of each endotoxin recognized by CD14, 13C 15N isotopically labeled Kdo2–Lipid A, a fully active chemically defined gram-negative endotoxin, and LTA lipid anchor, the minimal unit of LTA, were produced. This allowed detailed NMR spectral mapping of these agonist ligands bound to sCD14 which identified, for the first time, structural regions and features in each that are strongly affected during complex formation with sCD14. Additionally, the presence of differential dynamic behavior was seen in both CD14 and the ligands upon complexation. This behavior suggests a likely role for dynamics in the mechanism of pattern recognition by CD14, which uses the dynamic ability of specific residue combinations to differentially affect endotoxin binding. Using NMR, the dynamic behavior of CD14 was further investigated using temperature and pH-dependence studies of isotopically labeled CD14. These studies clearly demonstrated the presence of multiple conformations for several residues, and may provide a possible explanation for the broad specificity of ligand binding by CD14. In addition, the spin-labeling of isotopically labeled lipid A enabled the collection of intermolecular distances on CD14 bound lipid A.

abstract: Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- &Aring; wavelength, Bragg peaks were recorded to 8.5- &Aring; resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- &Aring; wavelength, and Bragg reflections were recorded to 3- &Aring; resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins. / Dissertation/Thesis / Ph.D. Chemistry 2011