Protein-protein interactions are of great importance to a number of essential biological processes including cell cycle regulation, cell-cell interactions, DNA replication, transcription and translation. Thus, an understanding of protein-protein interactions is critical for understanding many facets of cell function. Unfortunately, the tools and methods currently in use to identify and study protein-protein interactions focus largely on high affinity, stable interactions. However, the majority of the protein-protein interactions involved in regulatory processes have weak affinities and are transient in nature. Therefore, it is important to develop new biotechnology capable of detecting weak and/or transient protein-protein interactions in vivo. Here, we describe four new methods that allow for the identification and study of weak and/or transient protein-protein interactions in vivo. First, we developed a rapid method to convert Escherichia coli orthogonal tRNA/synthetase pairs into an orthogonal system for mammalian cells in order to site-specifically incorporate unnatural amino acids into any gene of interest using stop codon suppression. This method will allow the expression and purification of proteins that carry normally transient post-translational modifications. Second, we successfully employed site-specific unnatural amino acid incorporation to chemically cross-link a known homodimer, Sortase A, in vivo. Third, we developed a novel tetracycline repressor-based mammalian two-hybrid system and successfully detected homo- and hetero-dimers that are known to have weak binding constants. Finally, a synthetic antibody (termed a synbody) that binds weakly to the SH3 domain of the proto-oncogene Abelson tyrosine kinase was developed. The synbody can potentially be used as a first generation drug and/or biomarker. We hope that the methods developed in this dissertation will enable the scientific community to better understand weak/transient protein-protein interactions in vivo. / text

The tremendous increase in biological sequence data presents us with an opportunity to understand the molecular and cellular basis for cellular life. Comparative studies of these sequences have the potential, when applied with sufficient rigor, to decipher the structure, function, and evolution of cellular components. The accuracy and detail of these studies are directly proportional to the quality of these sequences alignments. Given the large number of sequences per family of interest, and the increasing number of families to study, improving the speed, accuracy and scalability of MSA is becoming an increasingly important task. In the past, much of interest has been on Global MSA. In recent years, the focus for MSA has shifted from global MSA to local MSA. Local MSA is being needed to align variable sequences from different families/species. In this dissertation, we developed two new algorithms for fast and scalable local MSA, a three-way-consistency-based MSA and a biclustering -based MSA. The first MSA algorithm is a three-way-Consistency-Based MSA (CBMSA). CBMSA applies alignment consistency heuristics in the form of a new three-way alignment to MSA. While three-way consistency approach is able to maintain the same time complexity as the traditional pairwise consistency approach, it provides more reliable consistency information and better alignment quality. We quantify the benefit of using three-way consistency as compared to pairwise consistency. We have also compared CBMSA to a suite of leading MSA programs and CBMSA consistently performs favorably. We also developed another new MSA algorithm, a biclustering-based MSA. Biclustering is a clustering method that simultaneously clusters both the domain and range of a relation. A challenge in MSA is that the alignment of sequences is often intended to reveal groups of conserved functional subsequences. Simultaneously, the grouping of the sequences can impact the alignment; precisely the kind of dual situation biclustering algorithms are intended to address. We define a representation of the MSA problem enabling the application of biclustering algorithms. We develop a computer program for local MSA, BlockMSA, that combines biclustering with divide-and-conquer. BlockMSA simultaneously finds groups of similar sequences and locally aligns subsequences within them. Further alignment is accomplished by dividing both the set of sequences and their contents. The net result is both a multiple sequence alignment and a hierarchical clustering of the sequences. BlockMSA was compared with a suite of leading MSA programs. With respect to quantitative measures of MSA, BlockMSA scores comparable to or better than the other leading MSA programs. With respect to biological validation of MSA, the other leading MSA programs lag BlockMSA in their ability to identify the most highly conserved regions.

Ribonuclease T1 oligonucleotide fingerprint (ONF) analysis was used to study genomic stability of La Crosse virus (Bunyaviridae) during vertical and horizontal transmission in the laboratory. No RNA genomic changes were detected in vertebrate cell culture-propagated virus isolated (following oral ingestion and replication) from the natural mosquito host, Aedes triseriatus. Genomic changes were not detected during transovarial passage of virus through two generations of mosquitoes or in virus isolated from suckling mice infected by transovarially infected mosquitoes. These results demonstrate that the La Crosse virus genome can remain relatively stable during transovarial transmission (TOT) in the insect host and during transfer between insect and vertebrate hosts. ONF analysis was used to demonstrate TOT of reassortant California serogroup bunyaviruses in Aedes treiseriatus. Mosquitoes were simultaneously inoculated with temperature sensitive mutants of La Crosse and Snowshoe hare viruses able to replicate at 33°C but not at 40°C. Putative reassortants, selected by replication at 40°C, were isolated from progeny mosquitoes and mice infected by these mosquitoes. ONF analysis confirmed that they were reassortants. Approximately 75% of the M segment and 25% of the L segment nucleotide sequences of Inkoo virus (Bunyaviridae) were determined by Sanger dideoxynucleotide sequencing of cDNA clones. Comparison of the M segment nucleotide and deduced amino acid sequences with those of four other bunyaviruses, representing two serogroups, revealed greater conservation of nucleotide than of amino acid sequence between serogroups. Areas of the sequences representing nonstructural protein(s) were less conserved than glycoprotein regions. The L segment nucleotide sequence begins with the known 3' end of the viral L segment and contains an open reading frame encoding the amino terminal 505 amino acids of the viral L protein. The amino acid sequence contains the glycine-aspartate-aspartate motif which is conserved in many RNA-dependent RNA polymerases. Comparison of the L segment sequences with those in the GEN Bank Data Base revealed no significant similarities with any other sequence.

Research presented in this dissertation falls into two parts: fragmentation mechanisms of peptide and fragmentation mechanism of amino acid derivatives. The study of peptide fragmentation may help to improve protein identification by incorporating the rules governing this process into search algorithms. This study elucidates the chemical 'rules' governing peptide dissociation. It is believed that these 'rules' can be incorporated into searching algorithms to achieve better protein identification. The present study focuses on the effects of different amino acids on fragmentation. Amino acids with a wide range of different chemical and physical properties are investigated, including amino acids with hydrophilic side chains, amino acids with aliphatic side chains and amino acids without side chains. It can be concluded from the present studies that the different amino acid properties have great influence on the peptide fragmentation and spectrum appearance.The study of fragmentation mechanisms of amino acid derivatives is another focus of this dissertation. Based on the fragmentation mechanism study, a quantification method was developed. The method can distinguish glutamine with 15N-label at N-terminal amine vs the side chain even if they have same molecular weight. Ammonia metabolism was successfully monitored by feeding mosquitoes with isotope-labeled compounds and subsequently measuring the amount of the labeled amino acids. This method demonstrates the power of mass spectrometry in metabolism studies.

The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.

The underlying nociceptive mechanisms in temporomandibular joint (TMJ) and masticatory muscles in many pain conditions are still unclear, largely due to the limited study of peripheral and central neural mechanisms affecting craniofacial musculoskeletal tissues. This study provided evidence in support of Hypothesis 1: Peripheral glutamatergic and capsaicin-sensitive mechanisms modulate the properties of primary afferents and brainstem neurons processing deep craniofacial nociceptive information. Effects of glutamate and capsaicin injected into the receptive field of deep craniofacial nociceptive afferents or TMJ of TMJ-responsive nociceptive neurons in trigeminal subnucleus caudalis/upper cervical cord (Vc/UCC) were studied in halothane-anesthetized rats. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to those of glutamate alone. When injected alone, glutamate and capsaicin also activated and induced central sensitization in most Vc/UCC neurons. Following glutamate injection, capsaicin evoked greater activity and less sensitization compared with capsaicin alone, whereas following capsaicin, glutamate was less effective in activating and sensitizing most Vc/UCC neurons. This apparent desensitizing effect of capsaicin on glutamate-evoked excitability of Vc/UCC neurons contrasts with the lack of capsaicin-induced modulation of glutamate-evoked afferent excitability, suggesting that peripheral and central sensitization may be differentially involved in the nociceptive effects of glutamate and capsaicin applied to deep craniofacial tissues. Further evidence of glutamate-capsaicin interactions was documented in the attenuation by TMJ pre-injection of glutamate receptor antagonists of jaw muscle activity reflexly evoked by TMJ injection of capsaicin. Moreover, additional findings support Hypothesis 2: Surgical cutaneous incision modulates the properties of brainstem neurons processing deep craniofacial nociceptive information. TMJ-responsive nociceptive Vc/UCC neurons could be activated by surgical incision of the skin overlying the TMJ and this incision-induced afferent barrage caused nociceptive neurons to be temporarily refractory to further capsaicin-induced central sensitization. These novel findings suggest that peripheral glutamate and capsaicin receptor mechanisms as well as surgical cutaneous incision may be involved in the nociceptive processing of deep craniofacial afferent inputs and may interact to modulate both activation as well as sensitization evoked from these tissues.

The underlying nociceptive mechanisms in temporomandibular joint (TMJ) and masticatory muscles in many pain conditions are still unclear, largely due to the limited study of peripheral and central neural mechanisms affecting craniofacial musculoskeletal tissues. This study provided evidence in support of Hypothesis 1: Peripheral glutamatergic and capsaicin-sensitive mechanisms modulate the properties of primary afferents and brainstem neurons processing deep craniofacial nociceptive information. Effects of glutamate and capsaicin injected into the receptive field of deep craniofacial nociceptive afferents or TMJ of TMJ-responsive nociceptive neurons in trigeminal subnucleus caudalis/upper cervical cord (Vc/UCC) were studied in halothane-anesthetized rats. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to those of glutamate alone. When injected alone, glutamate and capsaicin also activated and induced central sensitization in most Vc/UCC neurons. Following glutamate injection, capsaicin evoked greater activity and less sensitization compared with capsaicin alone, whereas following capsaicin, glutamate was less effective in activating and sensitizing most Vc/UCC neurons. This apparent desensitizing effect of capsaicin on glutamate-evoked excitability of Vc/UCC neurons contrasts with the lack of capsaicin-induced modulation of glutamate-evoked afferent excitability, suggesting that peripheral and central sensitization may be differentially involved in the nociceptive effects of glutamate and capsaicin applied to deep craniofacial tissues. Further evidence of glutamate-capsaicin interactions was documented in the attenuation by TMJ pre-injection of glutamate receptor antagonists of jaw muscle activity reflexly evoked by TMJ injection of capsaicin. Moreover, additional findings support Hypothesis 2: Surgical cutaneous incision modulates the properties of brainstem neurons processing deep craniofacial nociceptive information. TMJ-responsive nociceptive Vc/UCC neurons could be activated by surgical incision of the skin overlying the TMJ and this incision-induced afferent barrage caused nociceptive neurons to be temporarily refractory to further capsaicin-induced central sensitization. These novel findings suggest that peripheral glutamate and capsaicin receptor mechanisms as well as surgical cutaneous incision may be involved in the nociceptive processing of deep craniofacial afferent inputs and may interact to modulate both activation as well as sensitization evoked from these tissues.

Collagen fibrils are the main constituent of the extracellular matrix surrounding eukaryotic cells. Even though the assembly and structure of collagen fibrils is well characterized, very little is known about the physico-chemical properties of their surface which is one of the key determinants of their biological functions. One way to obtain surface sensitive structural and chemical data is to take advantage of the near field nature of surface and tip-enhanced Raman spectroscopy. Using Ag and Au nanoparticles bound to collagen type I fibrils, as well as tips coated with a Ag nanoparticles and a thin layer of Ag, we obtained Raman spectra characteristic of the first layer of collagen molecules at the surface of the fibrils. The most frequent Raman peaks were attributed to aromatic residues such as phenylalanine and tyrosine. We also observed in several instances Amide I bands with a full width at half maximum of 10-30 cm-1. The assignment of these Amide I bands positions suggests the presence of collagen-helices as well as alpha-helices and beta-sheets at the fibril’s surface. As a step towards in vivo characterization of collagen fibrils, fascicles removed from tendons were also examined with surface-enhanced Raman spectroscopy.

Approaches for the evaluation of plant protein ingredients for salmonid feeds were investigated in a series of four trials. The first trial compared the apparent digestibility coefficients (ADC) of crude protein (CP) and amino acids (AAs) of two novel products - Indian mustard protein concentrate (IMC, 62% CP) and Indian mustard protein meal (IMM, 42% CP), to a commercially available soy protein concentrate (SPC, 57% CP) for two salmonid species, rainbow trout and Atlantic salmon. The second trial involved assessment of relative bioavailability of arginine (Arg) from IMC, IMM and SPC compared to that of a crystalline Arg (L-Arg) in rainbow trout using slope-ratio assay. In the third trial, the effects of phytic acid (PA) and lignin on nutrient utilization and partitioning in rainbow trout were assessed. Finally, a series of experiments was conducted in the final trial to establish the evaluation criteria for pellet quality assessment. The ADC of CP and most AAs in IMC and IMM were high (>90%). Differences in the ADCs of some AAs can be attributed to the high PA intake by fish fed 30%-IMC diet. The significantly higher (P<0.05) bioavailability of Arg from IMC (123 to 187%) and IMM (116 to 211%) relative to that of L-Arg, as determined by various regression approaches, reaffirmed the findings of the first trial that these ingredients are of excellent protein quality and can readily be used in compounded fish feeds. It can be inferred from the lack of effects of PA, lignin or PA plus lignin on most indices of physiology, performance, and nutrient utilization in the pair-fed fish, that like any other animal, controls feed intake when in the presence of one or more dietary ANF. It was also shown in the pellet quality assessment trial that minor changes in dietary composition can significantly alter physical properties of aquaculture feed. This study highlighted the importance of a comprehensive assessment for the effective evaluation of the nutritive value of plant protein ingredients for use in aquaculture feeds. / Ontario Ministry of Natural Resources; BIOEXX Specialty Proteins Ltd.; Martin Mills Inc.; MITACS

The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].