Dissertations / Theses on the topic 'Antifreeze proteins'

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.<br>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.<br>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.

Antifreeze proteins from natural sources have been discovered to have cryoprotective function against freezing temperature, and have been tested for the application for cryopreservation of biological materials. However, none has been shown to match the effectiveness of current chemical cryoprotectants, such as dimethyl sulfoxide. One potential limitation with the application of antifreeze proteins is that they may only stay in the extracellular space around cells whereas chemical cryoprotectants can be penetrative. In this thesis project, we have designed, purified and explored the function of antifreeze proteins that were engineered with an intracellular delivery signal peptide, known as iPTD. We showed that iPTD-engineered antifreeze proteins had effective cell surface coverage within 30 minutes of incubation as shown by flow cytometry; however no intracellular protein delivery was observed under multiphoton microscopy. The plasma membrane was protected by iPTD-engineered antifreeze proteins during cryopreservation as seen in Calcein dye release assay, but cell recovery or proliferation was not observed after thawing. Given these properties of iPTD-engineered antifreeze proteins, we used them as red blood cell cryopreservation additives. By adding these modified antifreeze proteins, we were able to reduce the amount of glycerol (used for RBC cryopreservation) necessary to control freeze-induced hemolysis. Furthermore, the quality of thawed red blood cells is higher as protein addition resulted in high retention of intracellular ATP.<br>Medicine, Faculty of<br>Medicine, Department of<br>Graduate

This thesis describes the design, synthesis and measurement of ice growth inhibition properties of 16 analogues of the type I, a-helical protein from the winter flounder. This 37-residue protein has been proposed elsewhere to inhibit ice growth by a hydrogen bonding dominated mechanism in which the four threonine residues, that are equally spaced on one face of the helix, interact with the ice lattice. Four series of analogues were studied, incorporating systematic changes to the threonine residues, as well as other potential hydrogen bonding amino acids, and the helical conformation of the protein. In series 1 proteins, the central two threonine residues and all four threonine residues of the native protein were mutated to serine. In series 2 proteins two additional salt bridges (Lys7/Glu11 and Lys29/Glu33) were incorporated, and all four threonine residues in this sequence were mutated to valine, serine, alanine, 2-aminobutyric acid, isoleucine and glycine. In series 3 protein, the Asx residues (Asp 1, 5 and Asn 16, 27) were replaced by alanine and leucine, respectively. All four threonine residues in this sequence were mutated to valine, removing all potential hydrogen bonding amino acids. In series 4 analogues the role of helicity in the ice growth inhibition properties of these proteins was examined by stabilising, via covalent bridges, a truncated analogue of the native protein which showed reduced helicity. The solution conformation of all analogues was studied by circular dichroism spectroscopy, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. Circular dichroism studies showed that all analogues in series 1, 2 and 3 were essentially 100% ct-helical at low temperatures, except for the 2-aminobutyric acid and glycine analogues, which were estimated to be 85% and 70% helical, respectively. Circular dichroism showed that the amide bridged analogue in series 4 was able to increase the helicity of the truncated analogue by approximately 30%, and nuclear magnetic resonance spectroscopy provided further details regarding the residues within the sequence that adopted a highly (it—helical conformation. Analytical ultracentrifugation of the series 2 alanine, valine and threonine analogues showed that there was no aggregation or self-association occurring up to a concentration of 1 mM. Ice growth inhibition properties of the analogues were studied using nanoliter osmometry, the crystal habit test and the ice hemisphere test. The valine substituted analogue gave a distinct etching pattern in which the protein accumulated on the {2 O 5 1} plane of ice lb, and exhibited thermal hysteresis comparable to the native protein. The alanine and 2-aminobutyric acid substituted analogues both showed reduced hysteresis compared to the native protein. A distinct etch pattern in which the protein accumulated on the {2 l l 0} plane was observed for the alanine analogue. All other analogues showed no detectable hysteresis and no ability to modify ice growth. The results show that the threonine hydroxyl groups do not play a crucial role in the accumulation of the native protein at the ice/water interface and the inhibition of ice growth below the equilibrium melting temperature. The observed thermal hysteresis and ice growth inhibition of the valine, alanine and 2-aminobutyric acid analogues suggest that interactions between these hydrophobic sidechains and the growing ice crystals are more significant than in previously proposed models. The inability of series 3 analogues to inhibit ice growth indicates the importance of the polar Asx residues in the mechanism of action.

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...

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.

<p>In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are involved in these defence systems (Korsloot et al. 2004). Since an organism’s resources may be considered to be limited, the ability to restore homeostasis depends on the severity of the different forms of stress it experiences. It has been proposed that pollutants present in the environment may alter the ability to respond to climatic stressors like e.g. low temperature, desiccation (Holmstrup 2002).</p><p>This work deals with the possible consequences of combined stress from metal exposure and low temperature in cold hardy insects. Many of these insects produce so called <i>antifreeze proteins</i> that protect them from lethal freezing. Metallothioneins are metal binding proteins that are considered to be important in detoxification when animals are exposed to metals. Metallothioneins and most forms of antifreeze proteins from insects are known to contain unusually high amounts cysteine. Cysteine is considered to be semi-essential, since it must be derived from the essential amino acid methionine (Choen 2004). Induction of one of these two types of proteins may potentially deplete the cysteine pool and thus reduce the capacity to produce the other type. Alternatively, the animals might have evolved other structures to avoid a potential competition for cysteine. The purpose of the present work was to explore these possible scenarios.</p><br>Paper I and II reprinted with kind permission of Elsevier, Sciencedirect.com

In a quest to find novel methods to improve food preservation, food companies product pipelines are largely driven by the growing customers’ request for natural solutions. To date, freezing is still the most efficient method to preserve perishable foods, without altering substantially its taste and texture and avoid the use of preservatives. The uncontrolled growth of ice crystals, pose however a serious risk for the stability of many frozen goods, threatening their integrity and sensorial features. A paradigm is represented by ice cream, whose ice crystals microstructure is fundamental to impart its characteristic mouthfeel, and as such, need to be preserved. A step forward toward the improvement of techniques to prevent extensive ice crystals growth, was represent by the introduction, a few years ago, of AntiFreeze proteins also known as Ice Structuring Proteins (ISP). By directly interacting with the water molecules that make up the crystalline structure of ice, ISP disrupt the regularity of those planes and inhibit, to a certain extent, further addition of water molecules and consequent ice crystal growth. To date, the only case of application of ISP in commercial products, is that of a fish ISP produced by recombinant S. cerevisiae. Besides the technological advantages in ice cream manufacturing enabled by the use of this protein, being a GMO derived product hampered its diffusion in GMO-skeptical communities, like in Europe and other western countries. In this scenario, the aim of this doctoral project, in collaboration with the University of Padova with the support of Food Research & Innovation srl, was focused on finding novel ISP alternatives for preservation of frozen foods. The discovery of novel sources and methods to obtain ISP, were driven by two requirement that needed to be satisfied: 1) the ISP source had be perceived as “safe” from potential consumers, 2) the bioprocess to obtain those ISP compatible with industrial uses. On the basis of requirement n.1, we chose wheat as our preferred source of ISP and, after a thorough literature search about wheat ISP, we decided to start our investigation from a Triticum aestivum Thaumatin-Like Protein (TaTLP), for which the gene sequence was publicly available. The choice fell on TaTLP as this protein was previously recovered from wheat extracts, with the ability to inhibit ice recrystallization in ice cream. In contrast with data reported in literature for TaTLP isolated from the plant, the recombinant product didn’t show any ability to prevent ice crystals grow. After a thorough biochemical investigation, we concluded that the lack of activity wasn’t ascribable to incorrect processing or maturation of TaTLP, whose structural features resembled much that of other proteins belonging to the same family. Our hypothesis, is that TaTLP doesn’t exhibit any activity toward ice per se and that previous studies suggesting the opposite, may have derived from isolations of homologues TLP, that evolved ice binding domains. From our investigations on a wheat apoplastic extract containing ISPs, we found that the activity of those ISPs could be enhanced upon addition to the extract of exogenous proteins, not necessarily ISPs. At the moment, we don’t have strong experimental evidences to elucidate the mechanism at the molecular level, but we believe that some wheat ISPs, may have evolved a strategy to enhance their single contribution to the total ice growth inhibition activity of the plant. This enhancement may rely upon the interaction of wheat ISPs with other proteins, similarly to what was observed for AFP from the overwintering insect D. canadensis. In the search of a method to efficiently extract ISP from wheat that could be used as stabilizers of the ice phase in ice cream, we set up a one-step extraction process that involved high temperature treatment of the plant. This method represent a significant step forward in terms of simplicity and yield of ISPs, compared to previous extraction methods. Plus, the ISPs obtained thereof are heat resistant, an essential feature for proteins which are to be used in industrial foods. Wheat ISPs were then used to formulate ice cream in pilot pre-industrial plant; these ice cream were subjected to thermal shock to exacerbate temperature fluctuations experienced by ice cream in its path from the factory to the household freezers. The result is a stabilized ice cream, in which ice crystals growth was inhibited by the addition of a wheat extract containing ISPs. This extract could be considered as a natural ingredient, lacking the need to be declared as an additive and therefore not falling under the E-number labeling regulation for food ingredients. In parallel to the tests performed on ice cream, isolation and partial characterization of ISP active species from wheat extract were attempted. At the end of the purification process, comprising 3 consecutive chromatographic separations, 5 proteins were eluted in a single fraction showing activity towards ice crystals growth. Being that these proteins have very similar isoelectric points and electrophoretic mobility, we believe that they might be isoforms of the same species. To conclude, we designed a feasible process to obtain ISP from a vegetal source that successfully stabilized ice cream, this method having all the features required to be implemented on a larger scale for industrial production purposes. From the collateral results obtained from our investigations on wheat extracts, interesting clues about the nature of wheat ISP has emerged, as well and their possible roles in preventing ice growth in a physiological context. Those findings, contribute to broaden the knowledge of a category of ISP that is still poorly characterized and pose the basis for new research lines.<br>Nell’ambito della conservazione di prodotti alimentari, uno dei trend di mercato in continua espansione e che fonda le proprie basi sul costante incremento della domanda di mercato, riguarda lo sviluppo di soluzioni cosiddette “naturali”. Il congelamento è ad oggi l’unica tecnica di conservazione che consente di allungare la vita di cibi deperibili, senza alternarne sostanzialmente sapore e struttura e soprattutto senza ricorrere all’impiego di conservanti. La crescita incontrollata dei cristalli di ghiaccio pone un problema di stabilità del prodotto, che si ripercuote negativamente sulle proprietà qualitative dello stesso. Un caso emblematico è quello del gelato, in cui è indispensabile che la microstruttura di cristalli di ghiaccio venga mantenuta tale per garantirne la tipiche caratteristiche di palatabilità. Un contributo sostanziale alla tecnologia del controllo dei cristalli di ghiaccio avvenuto negli ultimi anni, è rappresentato dall’ introduzione delle cosiddette Proteine AntiFreeze, anche note come proteine Ice Structuring (ISP). Interagendo specificamente con le molecole d’acqua che costituiscono la struttura cristallina del ghiaccio, le ISP sono in grado di perturbarne la regolarità ed impedire, entro certi limiti, l’accrescimento incontrollato di cristalli di ghiaccio. L’unico esempio di ISP attualmente impiegata in ambito alimentare riguarda una proteina di pesce artico prodotta mediante tecnologia del DNA ricombinante con l’impiego di S. cerevisiae. Nonostante gli indubbi vantaggi e le possibilità che l’impiego di tale proteina ha consentito ai produttori di gelato, il suo successo in Europa e alcuni paesi occidentali è stato rallentato dalla diffidenza nei confronti dei prodotti a base di OGM. In questo scenario, alla base di questo progetto di ricerca, svolto nel contesto della scuola di dottorato presso l’Università degli Studi di Padova e grazie al contributo dell’azienda Food Research & Innovation srl, vi era la necessità di ricercare soluzioni alternative alle ISP già in commercio. Tali soluzioni dovevano necessariamente rispondere a due requisiti: 1) che le ISP venissero ricavate da fonti ritenute “affidabili” dal potenziale consumatore e 2) che il procedimento produttivo per l’ottenimento di tali ISP fosse compatibile con requisiti di applicabilità industriale. Sulla base del primo requisito, abbiamo scelto di utilizzare germogli di grano (T. aestivum) come fonte di ISP. Come prima cosa, a seguito di un’estensiva ricerca in letteratura delle ISP di grano, abbiamo scelto di partire con una Thaumatin-Like protein di Triticum aestivum (TaTLP), della quale era disponibile la sequenza genica. La nostra scelta è ricaduta su TaTLP, poiché tale proteina è stata ritrovata in estratti di grano con la capacità di inibire il processo di ricristallizzazione dei ghiaccio in campioni di gelato. Contrariamente a quanto riportato in letteratura sulla proteina isolata dalla pianta, la proteina ricombinante da noi ottenuta non ha alcun effetto sulla crescita dei cristalli di ghiaccio. A seguito di un’ approfondita caratterizzazione biochimica, abbiamo potuto stabilire che la mancata di attività non è da attribuire all’incorretta maturazione della proteina, che risulta strutturalmente e funzionalmente simile ad altre proteine appartenenti alla stessa classe. La nostra ipotesi è che la proteina in questione non abbia alcuna attività nei confronti dei cristalli di ghiaccio, e che gli studi precedenti si siano basati su proteine omologhe in cui tale attività è mediata da domini proteici specifici. A seguito di un indagine sperimentale su un estratto di ISP apoplastiche di grano, abbiamo scoperto che l’attività di tale proteine può essere incrementata, mediante l’aggiunta di proteine esogene all’estratto, non necessariamente ISP. Al momento, non siamo in possesso di solide evidenze sperimentali che ci consentano di validare un modello a livello molecolare. Tuttavia, riteniamo che alcune ISP di grano, possano aver evoluto un sistema per incrementare il proprio contributo, all’attività di prevenzione dalla crescita dei cristalli di ghiaccio a livello di organismo. L’incremento dell’attività delle ISP di grano potrebbe essere mediato dall’interazione diretta con altre proteine, in maniera simile a quanto riscontrato nel caso delle ISP ritrovate nelle larve dell’ insetto D. canadensis. Con lo scopo di identificare un processo per isolare le ISP dal grano da impiegare nella stabilizzazione del gelato, abbiamo messo a punto un procedimento di estrazione che consiste in una singola fase di estrazione a caldo dal materiale fogliare. Tale procedimento rappresenta un significativo miglioramento di processi estrattivi attualmente in uso, in ottica di semplicità del metodo e resa di ISP. Un ulteriore vantaggio è costituito dal fatto che al termine del processo, si ottengono ISP resistenti al calore, una caratteristica indispensabile per la loro applicabilità a cibi prodotti a livello industriale. Con le ISP ottenute in questo modo, sono stati preparati dei gelati in impianti pilota pre-industriali, i quali sono stati poi sottoposti a shock termico, per esasperare le fluttuazioni di temperatura che avvengono durante il ciclo di vita di un gelato, dal produttore al freezer del consumatore. Il risultato è un gelato stabilizzato in cui la crescita dei cristalli di ghiaccio dovuta allo stress termico è inibita mediante l’aggiunta di un estratto di grano come fonte di ISP. Tale estratto si configura come un ingrediente naturale, per il quale non è previsto un numero E con il quale vengono normalmente catalogati gli additivi alimentari. Parallelamente ai test sul gelato, abbiamo condotto una procedura di isolamento e caratterizzazione delle componenti ISP contenute dell’estratto. Al termine di un processo di purificazione che comprende 3 passaggi cromatografici, abbiamo ottenuto 5 specie proteiche che eluiscono in una singola frazione cromatografica ad alta attività. Date le simili proprietà di carica e peso molecolare verificate in gel bidimensionali, ipotizziamo che tali proteine siano isoforme di un’ unica specie. In conclusione, abbiamo definito un processo per l’ottenimento di ISP da fonte vegetale, compatibile con requisiti di produzione industriale ed efficacie nello stabilizzare il gelato. Dai risultati collaterali di questa ricerca sono emersi importanti informazioni in merito alla natura delle ISP di grano, così come spunti interessanti sul comportamento di queste proteine in un contesto fisiologico. Queste scoperte contribuiscono ad ampliare la conoscenza di una categoria di ISP sino ad oggi poco caratterizzata e pongono le basi per nuovi filoni di ricerca.

Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are proteins found in a wide variety of organisms adapted to survive in freezing temperatures. These proteins are powerful ice recrystallization inhibitors (IRI), slowing or even stopping ice crystal growth. This property is of significant interest in the area of cryopreservation due to ice recrystallization upon thawing being the limiting factor in the recovery of many biological materials. The need for reliable cryopreservation is becoming more and more necessary in many clinical areas, including regenerative and transplant medicine, and protein based therapeutics. The identification of new protein structures and sequences is crucially to understanding this ability. To this end l-type lectins, which show sequential homology to type 2 AFPs were explored and found to possess calcium dependant IRI activity. In addition, a secondary motif, amphipathicity was investigated. As many AFPs possess separate well defined hydrophilic/ hydrophobic domains this was thought to be a property of high importance. The antimicrobial peptide nisin A, which is amphipathic, was found to also possess cation dependant IRI activity. This identification demonstrates a new approach to identifying IRI active molecules and may further enhance our understanding of the mechanisms involved. As current “gold standard” methods for identification of IRI activity are relatively slow and time consuming signifying that the discovery of new IRI molecules is a slow process. In order to increase throughput of the discovery of novel IRI active molecules a gold nanoparticle assay was developed. Using this method serum proteins were discovered to possess weak but definite activity at higher concentrations. This is of particular interest as serum proteins such as fetal calf serum are commonly used in cryopreservation applications. The isolation of these remarkable proteins from primary sources in significant quantities is difficult and not financially viable, while recombinant expression is complicated by glycosylation of AFGPs. Furthermore non-native proteins may cause immunogenic problems. Therefore synthetic mimetic such as polymers and small molecules are highly appealing. Using a biomimetic approach, amphipathic metallohelicies with alpha helical character similar to that of a type 1 AFP were evaluated for ice recrystallization inhibition. It was found that these metallohelices could be optimized to completely inhibit ice recrystallization, demonstrating that by creating materials which mimic AFPs similar properties can be engineered successfully. In addition, polyampholyte polymers (polymers possessing both positive and negative side chains) were synthesized, and demonstrated to possess IRI activity when the ratio of positive to negative side chains was roughly 1:1. These were then used to significantly enhance post-thaw recovery rates of red blood cells (RBCs). Polymers are appealing as they are highly tunable and can be used to mimic proteins in a wide variety of biological areas. Polyampholyte polymers and previously identified IRI polymer; polyvinyl alcohol (PVA), added to polyethylene glycol were found to be a highly efficient way of preserving proteins and antibodies under freezing conditions. The application of polyampholytes and other IRI active molecules are therefore highly attractive in the cryopreservation of a whole range of biological materials, potentially yielding widespread clinical benefits.

Una proteina in grado di legare i cristalli di ghiaccio è definita proteina legante il ghiaccio o IBP acronimo dall’inglese ice-binding protein. Le IBP grazie alla loro capacità di abbassare il punto di congelamento dell’acqua, aumentando il gap di isteresi termica (TH). Questo intervallo è definito come la differenza tra il punto di fusione e di congelamento dell’acqua. La seconda attività delle IBP è l’inibizione della ricristallizzazione del ghiaccio (ice recrystallization inhibition, IRI). Infatti, queste proteine stabilizzano i piccoli cristalli di ghiaccio impedendo la formazione di cristalli di ghiaccio di grosse dimensioni che sono dannosi per le cellule. Le IBP sono state identificate in numerosi organismi tra cui pesci, insetti, batteri, alghe e lieviti. Queste proteine rappresentano un esempio di evoluzione convergente, infatti tutte le IBP condividono lo stesso meccanismo di legame con il ghiaccio nonostante una sorprendente diversità strutturale e funzionale. Questo lavoro di tesi è focalizzato sulla caratterizzazione funzionale e strutturale di EfcIBP, una IBP batterica identificata da analisi di metagenomica effettuate sul ciliato Antartico Euplotes focardii e sul consorzio batterico ad esso associato. La struttura 3D di EfcIBP è stata risolta mediante cristallografia ai raggi X e consiste in un β-solenoide con un α-elica parallela all’asse principale della proteina. L’analisi strutturale ha permesso di identificare tre diverse facce del solenoide denominate A, B e C. Simulazioni di docking suggeriscono che EfcIBP è in grado di legare i cristalli di ghiaccio tramite le facce B e C del solenoide. Questa ipotesi è stata verificata attraverso la progettazione razionale di 6 varianti che sono state prodotte e saggiate per la loro attività. In generale, questi risultati indicano che EfcIBP è in grado di legare i cristalli di ghiaccio attraverso le facce B e C del solenoide. Questa peculiarità strutturale si riflette in un’insolita combinazione di attività di IRI e TH. Infatti, EfcIBP presenta una notevole attività di IRI in un intervallo di concentrazione nanomolare e una attività di isteresi termica di 0.53°C alla concentrazione di 50 μM che la rende una IBP moderata. All’interno del gap di TH, i cristalli di ghiaccio presentano una forma esagonale, mentre a temperature al di sotto della temperatura di congelamento presentano una forma a “Saturno". La proteina chimerica formata dalla “green fluorescent protein” e da EfcIBP è stata utilizzata per determinare a quali piani del cristallo di ghiaccio la proteina è in grado di legarsi e con quale cinetica. I dati sperimentali suggeriscono che le peculiarità funzionali di EfcIBP sono dovute alla sua capacità di legare velocemente i piani basali e piramidali del cristallo di ghiaccio. Questi dati, insieme alla presenza di una sequenza segnale per la secrezione, suggeriscono che EfcIBP è secreta e svolge la funzione di mantenere liquido l’ambiente circostante aumentando lo spazio vitale. In conclusione, EfcIBP è un nuovo tipo di IBP con proprietà insolite di legame al ghiaccio e di attività di IRI. Questo studio ha contribuito ad identificare una nuova classe di IBP moderate che potrebbero essere sfruttate come crioprotettori in diversi settori come la criobiologia e quello alimentare.<br>Ice-binding proteins (IBPs) are characterized by the ability to control the growth of ice crystals. IBPs are active in increasing thermal hysteresis (TH) gap as they decrease the freezing point of water. On the other hand, IBPs can inhibit ice recrystallization (IRI) and stabilize small ice crystals at the expense of the harmful, large ones. IBPs have been identified in several organisms including higher Eukaryotes and microorganisms such as bacteria, yeasts and algae. Although IBPs share the ability to bind ice crystals, proteins from different sources present different 3D structures, from α-helix to β-solenoid proteins. This thesis is focused on the structural and functional characterization of EfcIBP, a bacterial IBP identified by metagenomic analysis of the Antarctic ciliate Euplotes focardii and the associated consortium of non-cultivable bacteria. The 3D structure of EfcIBP, solved by X-ray crystallography, consists in a β-solenoid with an α-helix aligned along the axis of the β-helix. It is possible to distinguish three different faces: A, B and C. Docking simulations suggest that B and C faces are involved in ice binding. This hypothesis was tested by the rational design of six variants that were produced and assayed for their activity. Overall, these experiments indicate that both solenoid faces contribute to the activity of EfcIBP. EfcIBP displays remarkable IRI activity at nanomolar concentration and a TH activity of 0.53°C at the concentration of 50 μM. The atypical combination between these two activities could stem from the ability of this protein to bind ice crystals through two faces of the solenoid. In the presence of EfcIBP, ice crystals show a hexagonal trapezohedron shape within the TH gap, and a unique “Saturn-shape” below the freezing point. A chimeric protein consisting of the fusion between EfcIBP and the green fluorescent protein was used to deeper investigate on this aspects by analyses of fluorescence ice plane affinity and binding kinetics. Overall, experimental data suggest that the EfcIBP unique pattern of ice growth and burst are due to its high rate of binding at the basal and the pyramidal near-basal planes of ice crystals. These data, together with the signal sequence for the secretion, suggest that EfcIBP is secreted in local environment where it becomes active in increasing the habitable space. In conclusion, EfcIBP is a new type of IBP with unusual properties of ice shaping and IRI activity. This study opens new scenarios in the field of IBPs by contributing to identify a new class of moderate IBPs potentially exploitable as cryoprotectants in several fields, such as cryobiology and food science.

Part I: Antifreeze Peptides Organisms living in icy environments produce antifreeze proteins to control ice growth and recrystallization. It has been proposed that these molecules pin the surface of ice crystals, thus inducing the formation of a curved surface that arrests crystal growth. Such proteins are very appealing for many potential applications in food industry, material science and cryoconservation of organs and tissues. Unfortunately, their structural complexity has seriously hampered their practical use, while efficient and accessible synthetic analogues are highly desirable. In the present work, we used molecular dynamics based techniques to model the interaction of three short antifreeze synthetic peptides with an ice surface. The employed protocols succeeded in reproducing the ice pinning action of antifreeze peptides and the consequent ice growth arrest, as well as in distinguishing between antifreeze and control peptides, for which no such effect was observed. Principal components analysis of peptides trajectories in different simulation settings permitted to highlight the main structural features associated to antifreeze activity. Modeling results are highly correlated with experimentally measured properties, and insights on ice-peptide interactions and on conformational patterns favoring antifreeze activity will prompt the design of new and improved antifreeze peptides. Part II: Molecular Similarity Molecular similarity is an important notion in chemistry, with applications in fields such as chemical databases and drug design. Molecular similarity is also important in chemical legislation, in particular in the evaluation process for orphan drugs (i.e., drugs for rare diseases). A new molecule needs to be dissimilar from any other existing drug for a given disease to be assigned the financially advantageous status of orphan drug. Currently, there are many ways to define whether two molecules are similar or dissimilar. Thus far, the European Medicines Agency has used majority voting on discretional judgments of similarity when assessing new drugs for rare diseases. Similarity in an inherently subjective concept, which depends on individual factors such as gender, age, state of mind, and previous experiences. Automated procedures that quantitatively and objectively evaluate molecular similarity are needed. Existing automated procedures are quite effective, but only take into account 2D molecular properties. We improved upon existing similarity-prediction procedures by including calculated 3D properties in the computational models. We created a new data-set of molecular similarity assessments, that includes complex and borderline similarity scenarios. We used the new data-set to test the existing procedures, and to build new and improved computational models.

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.<br>Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-07-18 15:24:42.082

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.

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.<br>Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-08-08 14:09:05.143

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.<br>Thesis (Master, Biochemistry) -- Queen's University, 2010-01-29 17:37:24.322

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.<br>Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-04-15 14:54:55.315

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.<br>Thesis (Master, Biology) -- Queen's University, 2009-09-01 10:04:00.72

Crystallization of water or water-encaged gas molecules occurs when nuclei reach a critical size. Certain antifreeze proteins (AFPs) can inhibit the growth of both of these, with most representations conceiving of an embryonic crystal with AFPs adsorbing to a preferred face, resulting in a higher kinetic barrier for molecule addition. We have examined AFP-mediated inhibition of ice and clathrate hydrate crystallization, and these observations can be both explained and modeled using this mechanism for AFP action. However, the remarkable ability of AFPs to eliminate „memory effect‟ (ME) or the faster reformation of clathrate hydrates after melting, prompted us to examine heterogeneous nucleation. The ubiquitous impurity, silica, served as a model nucleator hydrophilic surface. Quartz crystal microbalance-dissipation (QCM-D) experiments indicated that an active AFP was tightly adsorbed to the silica surface. In contrast, polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), two commercial hydrate kinetic inhibitors that do not eliminate ME, were not so tightly adsorbed. Significantly, a mutant AFP (with no activity toward ice) inhibited THF hydrate growth, but not ME. QCM-D analysis showed that adsorption of the mutant AFP was more similar to PVCap than the active AFP. Thus, although there is no evidence for „memory‟ in ice reformation, and the structures of ice and clathrate hydrate are distinct, the crystallization of ice and hydrates, and the elimination of the more rapid recrystallization of hydrates, can be mediated by the same proteins.

Cryoprotectants used commercially to minimize changes in texture and protein properties during freezing and frozen storage of fish typically impart an undesirable sweet taste. Since fish antifreeze proteins (AFP) are known to modify and suppress ice crystal growth, the objective of this study was to evaluate AFP as alternative cryoprotectants for frozen ling cod mince and natural actomyosin (NAM). Mince from ling cod was subjected to freeze-thaw abuse in the absence (control) or presence of AFP (5, 10, 50 or 500 ppm), AFP (50 ppm) with 0.3% phosphates, polyols (4% sucrose + 4% sorbitol or 8% trehalose), or AFP (50 ppm) with polyols (2% sucrose + 2% sorbitol). Freeze-thawed mince with AFP showed higher textural hardness, less salt extractable protein, and higher expressible moisture. These samples also formed a layer of ice crystals, which was not observed in polyol blends or control. Differential scanning calorimetry showed more unbound water in mince with AFP, while Raman spectroscopy indicated increased prevalence of β-sheet and random coil structures at the expense of α-helix. AFP failed to prevent loss of Ca-ATPase activity in NAM following freeze-thaw abuse. AFP solutions were evaluated at ambient and subzero (-0.5, -1.8, -4.0°C) temperatures using Raman spectroscopy. AFGP had small peaks near 1620 and 1674cm⁻¹ attributed to polyproline type-II helix and extended/unordered β-structures, respectively, and a strong band at 1070cm⁻¹ assigned to backbone C-C, C-N stretching and carbohydrate vibrations. Sharpening of the amide I band near 1645cm⁻¹ for AFPI at subzero temperatures showed strengthening of α-helix upon cooling. Strong hydrophobic interactions from aliphatic amino acids were seen at -0.5°C, and hydrogen-bonding and involvement of methyl groups were implicated at subzero temperatures. Frequency shifts in the O-H stretching band of water were observed in the presence of AFP at subzero temperatures. AFP did not prevent ice recrystallization or protein denaturation in fish mince during freeze-thawing. Conformational changes of AFP were observed at subzero temperatures, especially at -0.5°C. This information could be useful to study future applications of AFP in situations where intense ice crystallization formation will be desired or applications such as chemical adjuvants to cryosurgery.<br>Land and Food Systems, Faculty of<br>Graduate