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The unscheduled formation of gas hydrate plugs in oil and gas pipelines, which can lead to serious mechanical and personnel damage, is a problematic issue in the petroleum industry. Traditionally, thermodynamic inhibitors such as methanol have been used to control the formation of gas hydrates, but due to the large expenses and ecological risks associated with its use there is increased interest in the use of alternative hydrate inhibitors. They include kinetic inhibitors (KIs) and antiagglomerants (AAs) and as their names imply, function by interfering with the kinetics of hydrate formation and hydrate agglomeration. Recently, antifreeze proteins (AFPs) have shown to inhibit hydrates and have been proposed as hydrate inhibitors. Normally, AFPs function to protect the tissues of various organisms during freezing conditions. Initially they were found in polar fish, and were later recognized in insects, plants and microorganisms. AFPs are thought to function by lowering the freezing point of water through an adsorption-inhibition mechanism. This thesis has shown that antifreeze proteins (AFPs) are able to modify the crystal morphologies of structure II (sII) tetrahydrofuran (THF) similarly to the KI poly-N-vinylpyrrolidone (PVP) by adhering to the hydrate surface and inhibiting crystal growth. The AFPs were also tested on a high-pressure sII methane/ethane/propane hydrate and proved to have superior hydrate inhibition to PVP. Yet, the expense of purifying AFPs makes them impractical for industrial purposes, thus investigations into the use of cold-adapted bacteria as hydrate inhibitors proved that isolates capable of adsorbing to THF hydrate showed the most effective THF hydrate inhibition. These findings suggest a potential for the future development of biologically-based hydrate inhibitors. / Thesis (Master, Biology) -- Queen's University, 2009-09-01 10:04:00.72

The main success of my thesis has been to establish the mechanism by which antifreeze proteins (AFPs) bind irreversibly to ice crystals, and hence prevent their growth. AFPs organize ice-like water on their ice-binding site, which then merges and freezes with the quasi-liquid layer of ice. This was revealed from studying the exceptionally large (ca. 1.5-MDa) Ca 2+-dependent AFP from the Antarctic bacterium Marinomonas primoryensis (MpAFP). The 34-kDa antifreeze- active region of MpAFP was predicted to fold as a novel Ca 2+-binding β-helix. Site-directed mutagenesis confirmed the model and demonstrated that its ice-binding site (IBS) consisted of solvent-exposed Thr and Asx parallel arrays on the Ca 2+-binding turns. The X-ray crystal structure of the antifreeze region was solved to a resolution of 1.7 Å. Two of the four molecules within the unit cell of the crystal had portions of their IBSs freely exposed to solvent. Identical clathrate-like cages of water molecules were present on each IBS. These waters were organized by the hydrophobic effect and anchored to the protein via hydrogen bonds. They matched the spacing of water molecules in an ice lattice, demonstrating that anchored clathrate waters bind AFPs to ice. This mechanism was extended to other AFPs including the globular type III AFP from fishes. Site-directed mutagenesis and a modified ice-etching technique demonstrated this protein uses a compound ice-binding site, comprised of two flat and relatively hydrophobic surfaces, to bind at least two planes of ice. Reinvestigation of several crystal structures of type III AFP identified anchored clathrate waters on the solvent-exposed portion of its compound IBS that matched the spacing of waters on the primary prism plane of ice. Ice nucleation proteins (INPs), which can raise the temperature at which ice forms in solution to just slightly below 0oC, have the opposite effect to AFPs. A novel dimeric β-helical model was proposed for the INP produced by the bacterium Pseudomonas borealis. Molecular dynamics simulations showed that INPs are also capable of ordering water molecules into an ice- like lattice. However, their multimerization brings together sufficient ordered waters to form an ice nucleus and initiate freezing. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-08-08 14:09:05.143

Many techniques exist for protein analysis including gel electrophoresis, immunoassays and mass spectrometry to name a few. Although each of these techniques offers different advantages there are challenges associated with protein analysis that present limitations and drawbacks for each technique. These include limited sample volumes, incompatible matrices, complex mixtures, limit of detection, and complicated and time consuming analysis techniques. The research described in this thesis deals with methods which address the above mentioned challenges. The fabrication of enzymatic microreactors and the development of an extraction technique to remove proteins from incompatible matrices are presented. A low temperature solvent bonding technique was developed that can easily be modified to accommodate pH and temperature sensitive enzymes such as pepsin. Microfluidic chips fabricated with poly(methyl methacrylate) were constructed using this bonding method. The low temperature bonding method allows enzymes to be patterned on microfluidic devices prior to bonding with no negative impact on enzyme activity. Both peptic and tryptic reactors were fabricated using this technique. These devices have a lifetime of one month and can perform protein digestions in as little as 2.4 seconds. Alternatively, enzymatic microreactors were developed using a novel enzyme immobilization method on porous polymer monolith columns. These columns have the advantage of having the dual functionality of an enzymatic microreactor and an electrospray emitter permitting on-line digestions to be performed. Furthermore, the new immobilization method allows columns to have a longer lifetime as this method permitted the regeneration of the columns with fresh enzyme once enzyme activity was lost. To overcome the issues associated with incompatible matrices, an extraction technique using acetonitrile and octyl-β-D-glycopyranoside was developed to remove proteins from organic matrices. This was developed to detect the presence of prion proteins in biodiesel. Afterwards, the prion proteins were enzymatically digested and detected by mass spectrometry. Finally both types of enzyme microreactors were applied to proteins that are difficult to digest with traditional in-solution digestions. The digestion of prion proteins and α-1-protease inhibitor were found to be more efficient and conducted in significantly less time demonstrating the potential use of these devices in clinical research. / Thesis (Ph.D, Chemistry) -- Queen's University, 2011-10-02 19:11:35.576

Kinesins are motor proteins that convert chemical energy from ATP hydrolysis into mechanical energy used to generate force along microtubules, transporting organelles, vesicles, and proteins within the cell. Kar3 kinesins are microtubule minus-end-directed motors with pleiotropic functions in mating and mitosis of budding and fission yeast. In Saccharomyces cerevisiae, Kar3 is multifunctionalized by two non-catalytic companion proteins, Vik1 and Cik1. A Kar3-like kinesin and a single Vik1/Cik1 ortholog are also expressed by the filamentous fungus Ashbya gossypii, which exhibits different nuclear movement challenges and unique microtubule dynamics from its yeast relatives. We hypothesized that these differences in A. gossypii physiology could translate into interesting and novel differences in its versions of Kar3 and Vik1/Cik1. Presented here is a structural and functional analysis of recombinantly expressed and purified forms of these motor proteins. Compared to the previously published S. cerevisiae Kar3 motor domain structure (ScKar3MD), AgKar3MD displays differences in the conformation of the ATPase pocket. Perhaps it is not surprising then that we observed the maximal microtubule-stimulated ATPase rate (kcat) of AgKar3MD to be approximately 3-fold slower than ScKar3MD, and that the affinity of AgKar3MD for microtubules (Kd,MT) was lower than ScKar3MD. This may suggest that elements that compose the ATPase pocket and that participate in conformational changes required for efficient ATP hydrolysis or products release work differently for AgKar3 and ScKar3. There are also subtle structural differences in the disposition of the secondary structural elements in the small lobe (B1a, B1b, and B1c) at the edge of the motor domain of AgKar3 that may reflect the enhanced microtubule-depolymerization activity that we observed for this motor, or they could relate to its interactions with a different regulatory companion protein than its budding yeast counterpart. Although we were unable to gain experimentally determined high-resolution information of AgVik1, the results of Phyre2-based bioinformatics analyses may provide a structural explanation for the limited microtubule-binding activity we observed. These and other fundamental differences in AgKar3/Vik1 could explain divergent functionalities from the ScKar3/Vik1 and ScKar3/Cik1 motor assemblies. / Thesis (Master, Biochemistry) -- Queen's University, 2012-07-26 10:40:54.738

Genetic variants of milk proteins result from amino acid substitutions or small fragment deletion in the polypeptide chain. It is well documented that certain variants are closely related to milk production, milk composition and physico-chemical properties of milk such as heat stability and coagulation properties during cheesemaking. So far, all the genetic variants have been characterized by various electrophoretic methods. Therefore, only variants involving differences in net charges could be identified. Silent variants are the results of amino acid substitutions or deletions which do not accompany charge differences and hence remain undetected by conventional electrophoretic methods. The objective of the present project is to develop proper methodology to identify and characterize silent variants of milk proteins based on hydrophobic properties of amino acids. Individual caseins were isolated from 635 milk samples by anion-exchange chromatography and their electrophoretic phenotype was determined by polyacrylamide gel electrophoresis under alkaline and acidic conditions. Trypsin hydrolysis of alphas1-casein, beta-casein and kappa-casein followed by reversed-phase HPLC was performed to identify possible mutations causing changes in hydrophobicities of amino acids. Among 627 alphas1-casein BB, 415 kappa-casein AA, 158 beta-casein A1A1 and 128 beta-casein A2A 2 according to electrophoresis, it was possible to find 25, 11, 16 and 7 samples respectively as potential silent variants. Further analysis of the aberrant peptides from alphas1-casein BB, kappa-casein AA and beta-casein A2A2 by mass spectrometry did not confirm the existence of silent variants; whereas analysis of aberrant peptide from beta-casein A1A1 revealed a mutation resulting in an increase of 16 Da. Analysis of amino acid composition of this aberrant beta-casein A1A1 peptide 114--169 showed a Leu replacing a Pro residue. Results from amino acid sequencing confirmed this mutation to be located at pos