Antifreeze proteins (AFPs) help cold-adapted organisms survive below 0 ◦C by binding to and inhibiting the growth of ice crystals. In this way, AFPs depress the freezing point of aqueous fluids below the melting point of ice (thermal hysteresis; TH). They also have the ability to inhibit ice recrystallization in the frozen state (ice recrystallization inhibition; IRI). Some AFPs show an order of magnitude higher TH activity than others, and are termed ‘hyperactive’. One of the objectives of this thesis was to see if IRI activities of the hyperactive AFPs are also an order of magnitude higher than the moderately active AFPs. Using a capillary-based assay for IRI, the activities of three hyperactive and three moderately active AFPs were determined. There was no apparent correlation between hyperactivity in TH and high IRI activity. However, mutations of residues on the ice-binding face (IBF) of both types of AFP reduced IRI and TH activities to a similar extent. In this way, the use of IBF mutant AFPs showed that the IBF responsible for an AFP’s TH activity is also responsible for its IRI activity. Analysis of the diverse AFP structures solved to date indicate that their IBFs are relatively flat, occupy a significant proportion of the protein’s surface area and are more hydrophobic than other surfaces of the protein. The IBFs also often have repeating sequence motifs and tend to be rich in alanine and/or, threonine. The de novo design of an ice-binding protein was undertaken using these features to verify the underlying physicochemical requirements necessary for a protein’s interaction with ice. Using site-directed mutagenesis, a total of sixteen threonine substitutions were made on one of the four faces of a cyanobacterial protein with no endogenous TH activity. The inclusion of eight paired threonines on one face of this quadrilateral helix gave the engineered protein low levels of TH activity, but at the cost of destabilizing the structure to some extent. The results of this study have validated some of the properties needed for the ice-binding activity of AFPs. / Thesis (Master, Biochemistry) -- Queen's University, 2010-01-29 17:37:24.322

Advances in molecular biology have revolutionized the field of biotechnology and allowed the development of recombinant protein production as an alternative to harvesting proteins from their natural sources. Production of target proteins in controllable host organisms offers scalable and economic approaches to meet market needs. Current host cell expression systems vary, and each has advantages and disadvantages. Photoautotrophic organisms, like microalgae, represent alternatives to fermentative microbes with the promise of recombinant protein production from sustainable inputs like carbon dioxide as a carbon source. In this thesis, a Chlamydomonas reinhardtii strain that was recently developed for phototrophic high- density cultivation and nuclear transgene expression was used to express target recombinant proteins from its plastid genome as a demonstration of possibilities for expansion of its potential value. Here, sequences of two anti-freeze proteins, the insect Choristoneura fumiferana (CfAFP) and grass Lolium perenne ice binding protein (LpIBP), and a yellow fluorescent protein (YFP, mVenus) were adapted to algal chloroplast genome expression plasmids, transformed, and protein titers characterized under various nutrient and growth regimes in alga. Rather than antibiotic selection, transformants were selected based on photosynthesis restoration in a knock-out recipient strain. LpIBP and mVenus expression were detected by Western blot and in gel fluorescence and estimated to be expressed up to ~7.65% and ~8.41% total soluble protein, respectively, whereas expression of CfAFP was not observed in any transformant. This work forms the basis of further investigation of recombinant protein expression in C. reinhardtii in high-density antibiotic-free culture and may influence feasibility assessments of scale up processes.

An ice nucleating protein (INP) with 66% amino acid sequence identity to the better-known INP of Pseudomonas syringae has been described in an environmental isolate of P. borealis and designated InaPb. Despite the fact that INPs are classified as ice-binding proteins, InaPb showed little affinity for pre-formed ice and showed incorporation rates similar to Ina- strains. Additionally, it appeared to lack in the ability to shape ice or limit its growth. However, it was an effective ice nucleator. Using the coding sequence for InaPb and a green fluorescent protein tag (GFP), an InaPb-GFP fusion protein construct was inserted into a broad host expression vector in order to visualize the expression and localization of the protein in E. coli and an Ina- strain of P.syringae. The InaPb-GFP protein appears to localize at the poles of E. coli, but the nucleation temperature for these cells was only marginally above -9°C, which indicated poor nucleation activity. When expressed in Ina- P. syringae, the proteins showed clustering throughout the cell and an increased ability to nucleate ice following cold conditioning. The ability to nucleate ice was further increased by the removal of the GFP tag resulting in an average nucleation temperature more consistent with that seen in the native host P. borealis. Since inaPb transcript levels did not appear to change after cold conditioning, the clustering seen using fluorescence microscopy was likely the result of increased aggregation of protein in the membrane. Most INP- producing bacteria are associated with plant disease, but experiments with P. borealis suggested that the Ina+ phenotype was not indicative of pathogenicity in this strain. It is hoped that my contribution to the functional characterization of this INP will lead to a better understanding of these special proteins and their importance to the handful of bacteria that exhibit this activity. / Thesis (Master, Biology) -- Queen's University, 2012-05-15 09:55:52.506

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

<p> Four stucturally distinct types of macromolecular antifreezes have been previously isolated from the sera of polar marine fish. When the water temperature surrounding these organisms drops below -0.7°C, the freezing point of their bodily fluids, any contact with surrounding ice will nucleate internal ice crystal growth. The antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) synthesized by the fish act to inhibit the growth of existing ice crystals in their sera through direct adsorption to the ice lattice. The α-helical structure of type I AFP from winter flounder has been solved to atomic resolution and its mechanism of ice binding has been proposed. The NMR solution structure of a type III AFP from ocean pout has identified proteins in this class to exist in a β-sandwich conformation, however their mechanism of action remains uncertain.</p> <p> To facilitate the pursuit of an x-ray crystal structure solution, we subcloned the gene for a type III AFP (HPLC6) into pET15b and expressed recombinant His-rHPLC6 AFP in E. coli. Purified rmHPLC6 product has been successfully crystallized, and heavy atom soaks were performed in order to attempt a structure solution by multiple isomorphous replacement. The lone tyrosine in this recombinant AFP has been successfully derivatized in solution with iodine, and the modified protein was crystallized. In order to optimize the measurement of anomalous scattering information, modifications to our data collection system were required. Cryocrystallography techniques were employed to improve the quality of collected data.</p> <p> The expression, purification, crystallization and optimized data collection on an iodine-derivatized type III AFP from ocean pout will be presented here. This work has been instrumental in providing the high quality x-ray data required to solve the crystal structure to atomic resolution. Future examination of the solved structure will promote an increased understanding of the ice-binding mechanism exhibited by this class of proteins.</p> / Thesis / Master of Science (MSc)