Interpretation of crystallographic and mutational studies of antifreeze proteins (AFPs) requires molecular modeling of AFPs with ice. Most models proposed so far suggested H-bonds as the major driving force of AFP-ice association. However, the bulk water offers optimal network of H-bonds and van der Waals contacts to the isolated AFP and ice suggesting that corresponding components of free energy would not decrease upon AFP-ice association. In an attempt to resolve this controversy, we Monte Carlominimized complexes of several AFPs with taking into account, in addition to nonbonded interactions and H-bonds, the hydration potential for proteins (Augspurger and Scheraga, 1996). Parameters of the hydration potential for ice were developed basing on an assumption that at the melting temperature the free energy of water-ice association is small. Simulations demonstrate that desolvation of hydrophobic groups in the AFPs upon their fitting to the grooves at the ice surface presents the major stabilizing contributions to the free energy of AFP-ice binding. Our results explain available data on structure of AFPs and their mutational analyses, in particular, a paradoxical fact that substitution of Thr residues to Val does not affect potency of Winter Flounder AFP. / Thesis / Master of Science (MS)

Fishes of the perciform suborder Notothenioidei are found in Antarctic and sub-Antarctic waters that are separated by the Antarctic Polar Front (APF), with some species being distributed on both sides of this front. In this wide latitudinal range, these fishes are exposed to different temperatures ranging from -2 °C in the High Antarctic regions to 12 °C in the sub-Antarctic regions. To survive in icy Antarctic waters, the Antarctic notothenioid species have evolved antifreeze glycoproteins (AFGPs) that prevent their body fluids from freezing. The findings of past research on the AFGP attributes of several notothenioid species inhabiting ice-free sub-Antarctic environments have presented a complex picture. Furthermore, previous taxonomic studies split widely distributed notothenioids into different species and/or subspecies, with other studies disagreeing with these splits. To understand the response of the sub-Antarctic notothenioids to warmer, ice-free environments, it is necessary to have a good understanding of their antifreeze biology and systematics. Therefore, this study aimed to determine the association, if any, between the antifreeze attributes of sub-Antarctic notothenioid fishes and their taxonomic status. And more...

Winter rye produces ice-modifying antifreeze proteins upon cold treatment. Two of these antifreeze proteins are members of the large, highly conserved, ??-1,3-endoglucanase family. This project was designed to identify glucanase genes that are expressed during cold acclimation, wounding, pathogen infection, drought or treatment with the phytohormones ethylene and MeJa. Additionally, a more detailed proteomic analysis was to be carried out to evaluate the glucanase content of the apoplast of cold-acclimated (CA) winter rye. Results of 2D SDS-PAGE analysis revealed that non-acclimated whole leaf protein extracts contain at least two ??-1,3-endoglucanses while CA whole leaf protein extracts contain at least three ??-1,3-endoglucanses. Subsequent 2D SDS-PAGE analysis was conducted on the apoplast extracts of NA and CA winter rye plants revealed the limitations of standard 1D SDS-PAGE. The 2-dimensional gel analysis revealed that there is a minimum of 25 proteins within the apoplast of CA winter rye, including at least 5 ??-1,3-endoglucanases. Genome walking was used to isolate cold-responsive glucanase genes. The five genes isolated were designated scGlu6, scGlu9, scGlu10, scGlu11 and scGlu12. The cis-element pattern within the promoter of each gene was evaluated using online databases of documented plant cis elements. As expected, all of the promoters contained elements associated with cold, biotic and abiotic stresses, light regulation, and development. The expression patterns predicted by the cis elements in each promoter were compared to the mRNA abundance produced by each gene as detected by semi-quantitative reverse transcriptase PCR. In most cases, the abundance of transcripts arising from each gene loosely corresponded to the expression pattern predicted by the cis elements the corresponding promoter. Transcripts of scGlu9, 10 and 11 were present in cold-treated tissues and are candidates for ??-1,3-endoglucanases with antifreeze activity. The results presented in this thesis provide additional insight into the apoplast proteome of CA winter rye plants as well as the complexity of the signals controlling the proteins that reside there. Although there are still a number of unresolved questions, this research opens new directions for future studies in the cold acclimation process in winter rye and specifically for the contribution of ?? -1,3-endoglucanses.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 62-69.

The economic and safety hazards associated with the ability of gas hydrates to form in pipelines have prompted our interest in the inhibition of hydrate growth. Antifreeze proteins (AFPs) adsorb to ice surfaces and certain AFPs can also inhibit the growth of hydrates formed from water molecules organized in cage-like formations around a central small gas molecule. A Monte Carlo computational method for simulating the growth of ice crystals has been developed and it has proved useful in the understanding of the inhibition mechanism of these proteins. We have modified this crystal growth software in order to simulate the growth of large structure II gas hydrates, consisting of millions of water and gas molecules. This represents a first step towards investigating the effectiveness of novel compounds to inhibit hydrate growth in silico. Here, we describe these software modifications, and our efforts to incorporate type I AFP molecules into the hydrate growth simulations. Because both the docking interaction and inhibition mechanism for AFP towards hydrates remains unknown, we have set up a number of inhibitor screens to investigate possible AFP-hydrate docking models. Our goal is to reproduce the changes to gas hydrate morphology that have been observed in the presence of AFP, which will guide our choices for the binding alignment between AFPs and hydrates. This alignment will be instrumental for determining the AFPI-inhibition mechanism and should prove invaluable for the development of novel, hyperactive hydrate inhibitors.

The purpose of this study is to develop the production method of CO2 hydrate-slurry. In this paper, the production process of CO2 hydrates with pure water dissolved antifreeze proteins (AFPs) is discussed. CO2 hydrate-slurry can be transported from a production place to storage one with a small pressure loss. The AFPs have made the hydrate particles be small and well disperse. It is revealed that the Type III AFPs are effective for the inhibition of structure I hydrate production. By the present experiments, the induction time for the hydrate production increases, and moreover the formation rate of the hydrate and the increasing rate of an agitator torque decrease.

Antifreeze proteins (AFPs) are produced by a variety of organisms to either protect them from freezing or help them tolerate being frozen. Recent structural work has shown that AFPs bind to ice using ordered surface waters on a particular surface of the protein called the ice-binding site (IBS). These 'anchored clathrate' waters fuse to particular planes of an ice crystal and hence irreversibly bind the AFP to its ligand. An AFP isolated from the perennial ryegrass, Lolium perenne (LpAFP) was previously modelled as a right-handed beta helix with two proposed IBSs. Steric mutagenesis, where small side chains were replaced with larger ones, determined that only one of the putative IBSs was responsible for binding ice. The mutagenesis work also partly validated the fold of the computer-generated model of this AFP. In order to determine the structure of the protein, LpAFP was crystallized and solved to 1.4 Å resolution. The protein folds as an untwisted left-handed beta-helix, of opposite handedness to the model. The IBS identified by mutagenesis is remarkably flat, but less regular than the IBS of most other AFPs. Furthermore, several of the residues constituting the IBS are in multiple conformations. This irregularity may explain why LpAFP causes less thermal hysteresis than many other AFPs. Its imperfect IBS is also argued to be responsible for LpAFP's heightened ice-recrystallization inhibition activity. The structure of LpAFP is the first for a plant AFP and for a protein responsible for providing freeze tolerance rather than freeze resistance. To help understand what constitutes an IBS, a non-ice-binding homologue of type III AFP, sialic acid synthase (SAS), was engineered for ice binding. Point mutations were made to the germinal IBS of SAS to mimic key features seen in type III AFP. The crystal structures of some of the mutant proteins showed that the potential IBS became less charged and flatter as the mutations progressed, and ice affinity was gained. This proof-of-principle study highlights some of the difficulties in AFP engineering. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-07-18 15:24:42.082

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2000. / Typescript. Bibliography: leaves 74-79.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 160-171.