Cationic antimicrobial peptides (CAPs) offer a viable alternative to conventional antibiotics as they physically disrupt the bacterial membranes, leading to cell lysis and death. However, colonized bacteria often form “biofilms” – characterized by the overproduction of exopolysaccharides - that restrict the penetration of antibiotics; successful antimicrobial agents must evade this exopolysaccharide ‘matrix’ to reach the bacterial membrane. Since the Pseudomonas aeruginosa biofilm alginate traps CAPs by forming peptide-alginate complexes, the aim of this thesis is to better understand the mechanisms of interaction of CAPs with bacterial membranes and biofilm alginate. Using a series of CAPs designed in our lab derived from the sequence KKKKKK-AAFAAWAAFAA-NH2, we found that hydrophobicity, charge distribution, and amino acid composition of CAPs play important roles in their membrane disruptive power, bioactivities, alginate-binding and alginate-diffusion abilities. These findings suggest routes to an optimal balance of factors in CAP design to allow both biofilm penetration and bacterial membrane destruction.

Direct synthesis of aminoacyl-tRNA occurs using α-N-tBoc-protected aminoacyl phosphates and lanthanum salts. Deprotection of aminoacyl tRNA is essential prior to translation; however, the conditions of tBoc deprotection causes tRNA degradation. It was found that α-N-pentenoyl-protected aminoacyl phosphates, deprotected under mild conditions, are effectively used in lanthanum-mediated acylation of tRNA analogs. This provides an alternative route for aminoacyl-tRNA synthesis that maintains tRNA structure. Also, it was determined that α-N-deprotection of aminoacyl phosphates prior to aminoacylation still produces aminoacylated tRNA analogs. This establishes that acyl phosphates activate amino acids without inducing self-condensation, presumably due to electrostatic repulsion. Direct quantification of lanthanum-mediated tRNA aminoacylation was additionally undertaken utilizing a radiolabelled tRNA assay. From this, it was shown that lanthanum-mediated acylation does not promote deacylation and degradation of tRNA. These results have provided insight into lanthanum-mediated acylation of tRNA, ultimately allowing for use of the reagent in ribosomal translation.

Proper progression through the cell cycle is critical for cell growth and survival. Disruption of cell cycle progression can lead to cell cycle arrest and cell death. In addition, uncontrolled cell cycle progression can lead to cancer. Cells have evolved complex mechanisms to regulate each phase of the cell cycle to ensure proper cell cycle progression. In the presence of cellular stress, cells will respond promptly to arrest the cell cycle and allow repair. In order to study this complex process, it is important to identify the complete complement of proteins involved. I took two large-scale approaches to study the cell cycle. First, I down-regulated the majority of essential genes in Saccharomyces cerevisiae, and determined how depletion of individual gene product affected progression through the cell cycle. I determined that over 65% of essential genes I tested are most important at a specific cell cycle phase. In addition, I found that two genes, Smc4 and Cse1, have novel roles in S-phase of the cell cycle. In the second approach, I discovered two anti-proliferative compounds. Both compounds caused cell cycle delay in G1 phase of the cell cycle. Chemical genetic screens in yeast allowed me to determine the pathways most sensitive to each of these two compounds. By studying the response of cells to these compounds, I confirmed that compound 13 causes mitochondrial dysfunction in cells and compound 15 causes nuclear DNA damage. Furthermore, I found that both compounds are toxic in mammalian cells and that the responses that they elicit in mammalian cells are similar to those observed in yeast cells.

The intrinsically disordered nonphosphorylated and phosphorylated R region of CFTR and its interactions with NBD1 and SLC26A3 STAS have been characterized at residue-specific resolution, primarily using NMR. Limited chemical shift dispersion indicates that the R region is intrinsically disordered in solution and that no global folding event occurs upon phosphorylation. Chemical shifts of backbone nuclei and sidechain carbons were assigned. SSP values indicate that phosphorylation acts as a structural switch, with a reduction in helical propensity in multiple nonphosphorylated R region segments. Free nonphosphorylated and phosphorylated R region were characterized using a variety of structural probes. Fast timescale motion indicates the presence of structural contacts in many R region segments. Hydrodynamic radii are intermediate to those expected for fully folded or denatured proteins, with the phosphorylated R region being slightly more compact. The nonphosphorylated R region was further characterized, including measurements of molecular dimensions, N-H bond vector orientation and inter-residue distances from 6 spin label sites. Using these parameters as input to the program ENSEMBLE enabled calculation of a representative pool of nonphosphorylated R region conformations, indicating the presence of transient contacts that could not be directly discerned from the input data. Examining labeled R region with the addition of unlabeled NBD1 provided evidence that multiple segments of nonphosphorylated R region bind and are released from NBD1 with varying affinities in a highly dynamic equilibrium. Phosphorylation relieves these interactions, with the exception of limited R region interactions near S768 when NBD1 is ATP-bound. Largely similar nonphosphorylated R region residues bind both ATP-bound ΔF508 and wild-type NBD1. Addition of unlabeled R region to labeled ATP-bound NBD1 caused spectral changes indicative of a direct interaction with more than one surface or conformational changes within NBD1 that are transmitted from one binding surface to other surface(s). Binding of unlabeled SLC26A3 STAS domain to labeled phosphorylated R region was also monitored and indicated that similar R region segments bind NBD1 and STAS, suggesting a direct competition between these two domains for binding. A model is proposed where the R region acts as a regulatory hub, integrating interactions with a variety of partners to regulate channel function.

Vts1 is a member of the Smaug protein family, a group of sequence-specific RNA-binding proteins that regulate mRNA translation and degradation by binding to consensus stem-loop structures in target mRNAs. Using RNA reporters that recapitulate Vts1-mediated decay in vivo as well as endogenous mRNA transcripts, I show that Vts1 regulates the degradation of target mRNAs in Saccharomyces cerevisiae. In Chapter Two, I focus on the mechanism of Vts1-mediated mRNA decay. I demonstrate that Vts1 initiates mRNA degradation through deadenylation mediated by the Ccr4-Pop2-Not deadenylase complex. I also show that Vts1 interacts with the Ccr4-Pop2-Not deadenylase complex suggesting that Vts1 recruits the deadenylase machinery to target mRNAs, resulting in transcript decay. Following poly(A) tail removal, Vts1 target transcripts are decapped and subsequently degraded by the 5’-to-3’ exonuclease Xrn1. Taken together these data suggest a mechanism of mRNA degradation that involves recruitment of the Ccr4-Pop2-Not deadenylase to target mRNAs. Previous work in Drosophila melanogaster demonstrated that Smg interacts with the Ccr4-Pop2-Not complex to regulate mRNA stability, suggesting Smaug family members employ a conserved mechanism of mRNA decay. In Drosophila, Smg also regulates mRNA translation through a separate mechanism involving the eIF4E-binding protein Cup. In Chapter Three, I identify the eIF4E-associated protein Eap1 as a component of Vts1-mediated mRNA decay in yeast. Interestingly Cup and Eap1 share no significant homology outside of the seven amino acid eIF4E-binding motif. In eap1 cells mRNAs accumulate as deadenylated capped species, suggesting that Eap1 stimulates mRNA decapping. I demonstrate that the Eap1 eIF4E-binding motif is required for efficient degradation of Vts1 target mRNAs and that this motif enables Eap1 to mediate an interaction between Vts1 and eIF4E. Together these data suggest Vts1 influences multiple steps in the mRNA decay pathway through interactions with the Ccr4-Pop2-Not deadenylase and the decapping activator Eap1.

Large multi-component structures play an essential role in many crucial cellular processes. The morphogenetic pathway of the long, non-contractile tail of bacteriophage λ provides a superb paradigm for studying the assembly of macromolecular complexes. This thesis describes the structural and functional characterization of two λ tail proteins, gpU and gpV, with the aim of improving our understanding of phage tail assembly and evolution, while also providing a starting point to answering some of the fundamental questions surrounding the assembly and function of other supramolecular structures. Tail Terminator Proteins (TrPs) play an essential role in regulating the length of phage tails, and serve as the interaction surface for phage heads. To provide insight into the mechanisms by which TrPs exert their functions, I have determined the X-ray crystal structure of gpU, the TrP from phage λ, in its biologically relevant hexameric state. The gpU hexamer displays several flexible loops that are involved in head and tail binding. By comparing the hexameric crystal structure of gpU to its previously determined NMR solution structure I was able to identify large structural rearrangements in the protein, which are likely induced upon oligomerization. In addition, I have shown that the hexameric structure of gpU is very similar to the structure of a putative TrP from a contractile phage tail even though they display no detectable sequence similarity. This finding implies that the TrPs of non-contractile tailed phages are evolutionarily related to those of contractile-tailed phages. To determine the mechanism by which tail tubes self-assemble prior to termination, I have determined the NMR solution structure of the N-terminal domain of gpV (gpVN), the protein comprising the major portion of the phage λ tail tube. I found that approximately 30% of gpVN is disordered in solution and that some of these disordered regions are biologically important. Intriguingly, my gpVN structure is very similar to a previously solved tail tube protein from a contractile-tailed phage, once again suggesting an evolutionary connection between these two distinct tail types. A remarkable structural similarity is also seen to the hexameric structure of Hcp1, a component of the bacterial type VI secretion system. This finding, coupled with other similarities between phage and type VI secretion proteins support an evolutionary relationship between these systems. Using Hcp1 as a model, I proposed a mechanism for the oligomerization and polymerization of gpV involving several disorder-to-order transitions. Further supporting the importance of unstructured regions, I have shown that the unstructured linker between the N- and C-terminal domains of gpV is crucial for protein function and that a complete truncation of the C-terminal domain (gpVC) results in a 100-fold decrease in activity compared to full-length gpV (gpVFL). To provide insight into the role of gpVC, I determined its NMR solution structure and showed that it possesses an Ig-like fold, however the function of gpVC remains unknown. Interestingly, the gpVC structure revealed the location of two residues that when mutated were previously shown to either abrogate (G222D) or restore (G222D/P227L) function of gpVFL. In addition to being inactive, I demonstrated that the G222D mutation also exerts a temperature dependent dominant negative phenotype. My preliminary NMR data suggests that G222D causes gpVC to partially unfold and that this destabilized form of the domain interacts with gpVN in a region that is likely involved in both oligomerization and hexamer-hexamer interactions. To further our understanding of how these mutations exert their effect, I determined the NMR solution structure of gpVC-P227L. My structure reveals that the β7-β8 region of gpVC-P227L is altered compared to gpVC-WT and suggests that the conformational changes in gpVC-P227L may protect the domain from protein-folding defects induced by the G222D mutation.

Leukocyte type core 2 {beta}1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Gal{beta}1,3[GlcNAc{beta}1,6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Gal{beta}1,3GalNAc-O-Ser/Thr), respectively. The primary objective of the work described in this thesis is to shed light on the structure, catalytic mechanism and substrate specificity of C2GnT-L. Since glycosyltransferases are membrane bound glycoproteins that possess disulphide bonds, the first challenge was to produce sufficient quantities of C2GnT-L for biochemical and structural characterization. To this end, C2GnT-L and various active site mutants were expressed and purified from stably transformed mammalian cell lines. The x-ray crystal structure of wild-type C2GnT-L was solved, in both its apo and acceptor substrate complexed forms. The structures, along with revealing the structural basis for acceptor substrate specificity, showed that C2GnT-L belongs to the GT-A glycosyltransferase fold type. This was a surprising result given that, to this day, C2GnT-L is the only GT-A glycosyltransferase that does not possess a DXD motif and does not require a divalent metal ion for catalysis. The mechanism of the metal ion-independent C2GnT-L activity, within the context of the metal ion-dependent GT-A fold, was probed further using site directed mutagenesis in conjuncition with x-ray crystallography, enzyme assays, and frontal affinity chromatography. It was found that positively charged side chains in C2GnT-L functionally replace the divalent metal ion found in other GT-A glycosyltransferases, providing evidence for a convergence of metal ion-independent activity between GT-A and GT-B glycosyltransferase fold types.

Sliding clamps and their cognate clamp loaders facilitate DNA synthesis, DNA repair, and sister chromatid cohesion in eukaryotes. ELG1 (enhanced level of genome instability) encodes a member of the fourth clamp-loader-like complex identified to date, and is important in the maintenance of genome integrity. Like all clamp loaders, Elg1 is a replication factor C (RFC) homologue. I examined the roles of the unique and conserved regions of S.cerevisiae Elg1 in resistance to exogenous DNA damage and suppression of spontaneous DNA damage. The conserved RFC region of Elg1 mediates association with chromatin function. The unique C- terminus of Elg1 mediates oligomerization with Rfc2-5, a core complex present in all clamp loaders, and is essential for Elg1 function. Finally, the N-terminus of Elg1 promotes its nuclear localization and contributes to the maintenance of genome stability. The Elg1-RFC complex most likely functions in collaboration with the sliding clamp PCNA. Combining mutations in ELG1 and PCNA results in endogenous DNA damage, which activates a noncanonical DNA damage response that results in upregulation of dNTP production. Increased dNTP pools allow significant DNA synthesis to occur at hydroxyurea (HU) concentrations that prevent replication in wild type cells. However, consistent with the recognized correlation between dNTP levels and spontaneous mutation, the double mutant exhibits a significant increase in mutation frequency. These phenotypes are also detectable in the single mutants although to a lesser extent. Together, these findings suggest that spontaneous mutagenesis stimulated by endogenous DNA damage may be a general feature of the DNA damage response.

When prions are transmitted between species, there can be a delay in pathogenesis due to a phenomenon referred to as the transmission barrier. Some species also show very low susceptibility to prion disease. In this study I hypothesized that the susceptibility of species to prion disease is proportional to the tendency of their endogenous prion protein, PrP, to adopt the β-state, an oligomeric form of misfolded recombinant PrP that is rich in β sheet. Using a novel method of two-wavelength CD analysis, it could be shown that recombinant PrP from prion-susceptible species have a higher propensity to refold to the β-state than resistant species. The crystal structure of rabbit PrPC 121-230 revealed a helix-cap motif at the N-terminus of helix-2 that contributes to the reduced β-state propensity of rabbit PrP. Single amino acid changes in the sequence of PrP can lead to a transmission barrier and/or resistance in species. Mutating single residues in rabbit PrPC to those found in corresponding positions in hamster PrPC, ablated the helix-cap observed in the wild-type and caused an increase in the β-state propensity of rabbit PrP. Conversely, a decrease in β-state propensity was observed when rabbit mutations were introduced into PrP of hamster, a susceptible species. A dimeric association is hypothesized to be involved in the function of PrP and/or the conversion mechanism to infectious prion. In the structures of the wild-type and mutant rabbit PrPCs a dimeric arrangement was observed in the asymmetric unit of the crystals. Using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), a dimer of rabbit and hamster PrPC was crosslinked in solution. The dimer crosslink was specific and dependent on the tertiary structure of PrPC. Crosslinking of the β-state octamer with EDC showed that similar contacts may be present in this oligomeric form. Together these data provide strong evidence that species susceptibility is linked to β-state propensity.

Systematic identification of protein-protein interactions (PPIs) on a genome scale has become an important focus of biology, as the majority of cellular functions are mediated by these interactions. Several high throughput experimental techniques have emerged as effective tools for querying the protein-protein interactome and can be broadly categorized into those that detect direct, physical protein-protein interactions and those that yield information on the composition of protein complexes. Tandem affinity purification followed by mass spectrometry (TAP/MS) is an example of the latter that identifies proteins that co-purify with a given tagged query (bait) protein. Though TAP/MS enables these co-complexed associations to be identified on a proteome scale, the amount of data generated by the systematic querying of thousands of proteins can be extremely large. Data from multiple purifications are combined to form a very large network of proteins linked by edges whenever the corresponding pairs might form an association. Only a fraction of these pairwise associations correspond to physical interactions, however, and further computational analysis is necessary to filter out non-specific associations. This thesis examines how differing computational procedures for the analysis of TAP/MS data can affect the final PPI network, and outlines a procedure to accurately identify protein complexes from data consolidated from multiple proteome-scale TAP/MS experiments in the budding yeast \textit{Saccharomyces cerevisiae}. In collaboration with the Greenblatt and Emili laboratories at the University of Toronto, this methodology was extended to yeast membrane proteins to derive a comprehensive network of 13,343 PPIs and 720 protein complexes spanning both membrane and non-membrane proteins.