No description available.

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

Antifreeze proteins (AFPs) protect organisms from freezing damage at subzero temperatures. They do this by adsorbing to the surface of nascent ice crystals to block further ice growth. The key property of AFPs is to be soluble in liquid water but bind irreversibly to water in the solid state. Hypotheses for the mechanism by which AFPs recognize and bind ice have gone through several radical revisions without a consensus emerging. The remarkable diversity of independently evolved AFP structures, the multiple ice planes bound by AFPs, and uncertainty about the location of the ice-binding site(s) have all added to the difficulty of deducing a unified mechanism of AFP action. The central thesis of my research is that the characterization of additional AFPs will elucidate rather than obfuscate the mechanism of action. To this end I have advanced knowledge about three hyperactive AFPs. A reliable protocol to express and purify a sufficient quantity of type I hyperactive AFP was developed for further characterization studies. Initial crystallization trials using the recombinant material have produced consistent crystals for diffraction and resolution. A model of the recently discovered snow flea AFP was generated via de novo methods. The folding scheme is polyproline type II helices stacked into anti-parallel sheets, which was to our knowledge previously unobserved in monomeric proteins. The model was subsequently confirmed to be within 1 Å accuracy by X-ray crystallography performed by another group. I have also screened several insects for antifreeze activity. By using mass-spectrometry sequencing and a cDNA library, novel AFPs (3 kDa and 8kDa) were discovered from overwintering inchworms. The translated proteins were subsequently de novo modelled. After a thorough analysis of the literature, I reason that conflicting results from various AFP studies can be resolved. The hydrogen-bond ice-binding hypothesis was re-introduced to work coherently with elements of the hydrophobic ice-binding theory. We have proposed a unifying mechanism termed “anchored clathrate water,” which is supported by the water bonding on ice-binding surfaces reported both in in silico and in NMR studies. The new data I have obtained have further reinforced and expanded the hypothesis. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-04-15 14:54:55.315

The formation of gas hydrates in the oil and gas industry causes numerous problems that require costly solutions and operation downtime. A great deal of hydrate research has focused on their prevention either through kinetic or thermodynamic inhibitors. Recently, antifreeze proteins (AFPs) produced by cold adapted organisms have been found to have a kinetic inhibitory effect on clathrate hydrates. / Kinetic experiments were conducted on the methane-water system in the presence of AFPs by measuring the gas uptake during the formation of methane hydrate in a 610 cc high pressure crystallizer. These experiments were performed at temperatures ranging from 277.15 K to 280.65 K, pressures of 5800 KPa to 8100 KPa and at an AFP concentration of 0.01 mM. / The results of these experiments showed that the presence of AFPs affect methane hydrate formation in multiple ways. They were shown to increase the nucleation time, reduce the initial growth rate of methane hydrate at the time of nucleation and there was evidence to suggest that they also have an anti-agglomerating effect on hydrate crystals.

No description available.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Structure-function relationships for an alanine-rich, α-helical antifreeze polypeptide (AFP) from winter flounder were studied with the goal of understanding how AFPs depress the freezing point of water. A 37-residue native AFP and 23 analogs with systematic variations in the polypeptide chain were synthesized, and the α-helix content, antifreeze activity, and effect on growth rates of ice crystals along the a and c axes were determined. The results indicate that both the regularly spaced threonine and asparagine (or aspartic acid) residues are critical for maximal activity, and that the asymmetric arrangement of these residues on the helix face causes asymmetric adsorption of AFPs on the ice surface. Charged-residues, except for C-terminal Arg, are not very critical for antifreeze activity. Studies of hydrophobic residue mutants showed that the overall hydrophobicity is not particularly important. However, the Ala residue in position 17 appears to be important, because replacement with a bulky group abolishes antifreeze activity, presumably by interfering with the favorable side-to-side hydrophobic A model for binding of the winter flounder AFP to ice is proposed, whereby the AFP inhibits the growth of ice crystals by hydrogen bonding of Thr, Asn and Asp side chains in a specific pattern to the { 20 21 } hexagonal bipyramidal planes of ice, unidirectionally along the vector <1102>. It is further proposed that ice crystal growth inhibition occurs by a two-step mechanism: first individual AFP molecules hydrogen bond to ice reversibly, allowing slow growth of ice crystal; then at sufficiently high AFP concentrations, the AFP molecules begin to pack together on the binding surface by cooperative, side-to-side, hydrophobic interpeptide interactions, resulting in essentially irreversible binding and arrested ice crystal growth. The D-enantiomer of the AFP was also synthesized. The D and L-enantiomers alone, as well as a 50:50 mixture of D and L, all show identical antifreeze activity. These results indicate that complete coverage of the ice surface is not necessary, and suggest a model whereby AFP molecules bind in patches on the ice surface. / 2999-01-01

No description available.

Thesis (M.S.)--Ohio University, November, 2007. / Title from PDF t.p. Includes bibliographical references.

Thesis (M.S.)--Ohio University, August, 2004. / Title from PDF t.p. Includes bibliographical references (leaves 116-119).