Thermal Hysteresis Activity Research Articles

Evaluation of Thermal Hysteresis Activity of Ice-binding Proteins Using Ice-etching and Molecular Docking

Evaluation of Thermal Hysteresis Activity of Ice-binding Proteins Using Ice-etching and Molecular Docking

Identification and Characterization of an Isoform Antifreeze Protein from the Antarctic Marine Diatom, Chaetoceros neogracile and Suggestion of the Core Region.

Antifreeze proteins (AFPs) protecting the cells against freezing are produced in response to extremely low temperatures in diverse psychrophilic organisms, and they are encoded by multiple gene families. The AFP of Antarctic marine diatom Chaetoceros neogracile is reported in our previous research, but like other microalgae, was considered to probably have additional genes coding AFPs. In this paper, we reported the cloning and characterization of additional AFP gene from C. neogracile (Cn-isoAFP). Cn-isoAFP protein is 74.6% identical to the previously reported Cn-AFP. The promoter sequence of Cn-isoAFP contains environmental stress responsive elements for cold, thermal, and high light conditions. Cn-isoAFP transcription levels increased dramatically when cells were exposed to freezing (−20 °C), thermal (10 °C), or high light (600 μmol photon m−2 s−1) stresses. The thermal hysteresis (TH) activity of recombinant Cn-isoAFP was 0.8 °C at a protein concentration of 5 mg/mL. Results from homology modeling and TH activity analysis of site-directed mutant proteins elucidated AFP mechanism to be a result of flatness of B-face maintained via hydrophobic interactions.

Molecular Insight into the Adsorption of Spruce Budworm Antifreeze Protein to an Ice Surface: A Clathrate-Mediated Recognition Mechanism.

The principal mechanism of ice recognition by antifreeze protein (AFP) has been a topic of intense discussion in recent times. Despite many experimental and theoretical studies, the detailed understanding of the process remains elusive. The present work aims to explore the molecular mechanism of ice recognition by an insect AFP from the spruce budworm, sbwAFP. As evident from our simulation, the water dynamics becomes very sluggish around the ice binding surface (IBS) as a result of the combined effect of confinement and ordering induced by the perfectly aligned methyl side chains of threonine residues, the THR ladder. The hydroxyl groups of threonine form strong hydrogen bonds with few of those highly ordered water molecules that are close to the THR ladder, which is the origin of anchored clathrate water at the IBS of sbwAFP. We propose anchored clathrate-mediated basal plane recognition by sbwAFP. The AFP adsorbed on the basal plane through water clathrate framed around the IBS. The surface of the basal plane and anchored clathrate water completes the caging around the threonine residues, which is the origin of the binding plane specificity of sbwAFP. This adsorbed AFP-ice complex undergoes dynamic crossover to a hydrogen-bonded complex within the thermal hysteresis (TH) regime of this particular AFP. The anchored clathrate water becomes part of the newly grown basal front as a result of the geometrical matches between the basal plane and the anchored clathrate water repeat distance. This observation provides a structural rationale for the experimentally observed time-dependent increase in TH activity for insect AFP. Our study proposes clathrate-mediated ice recognition by AFP and elucidates the dynamic events involved during ice binding by the insect AFP.

Oxidized Quasi-Carbon Nitride Quantum Dots Inhibit Ice Growth.

Antifreeze proteins (AFPs), a type of high-efficiency but expensive and often unstable biological antifreeze, have stimulated substantial interest in the search for synthetic mimics. However, only a few reported AFP mimics display thermal hysteresis, and general criteria for the design of AFP mimics remain unknown. Herein, oxidized quasi-carbon nitride quantum dots (OQCNs) are synthesized through an up-scalable bottom-up approach. They exhibit thermal-hysteresis activity, an ice-crystal shaping effect, and activity on ice-recrystallization inhibition. In the cryopreservation of sheep red blood cells, OQCNs improve cell recovery to more than twice that obtained by using a commercial cryoprotectant (hydroxyethyl starch) without the addition of any organic solvents. It is shown experimentally that OQCNs preferably bind onto the ice-crystal surface, which leads to the inhibition of ice-crystal growth due to the Kelvin effect. Further analysis reveals that the match of the distance between two neighboring tertiary N atoms on OQCNs with the repeated spacing of O atoms along the c-axis on the primary prism plane of ice lattice is critical for OQCNs to bind preferentially on ice crystals. Here, the application of graphitic carbon nitride derivatives for cryopreservation is reported for the first time.

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a 'splat assay', which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called 'ice-affinity purification'. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

Cryo-protective effect of an ice-binding protein derived from Antarctic bacteria.

Cold environments are populated by organisms able to contravene deleterious effects of low temperature by diverse adaptive strategies, including the production of ice binding proteins (IBPs) that inhibit the growth of ice crystals inside and outside cells. We describe the properties of such a protein (EfcIBP) identified in the metagenome of an Antarctic biological consortium composed of the ciliate Euplotes focardii and psychrophilic non-cultured bacteria. Recombinant EfcIBP can resist freezing without any conformational damage and is moderately heat stable, with a midpoint temperature of 66.4 °C. Tested for its effects on ice, EfcIBP shows an unusual combination of properties not reported in other bacterial IBPs. First, it is one of the best-performing IBPs described to date in the inhibition of ice recrystallization, with effective concentrations in the nanomolar range. Moreover, EfcIBP has thermal hysteresis activity (0.53 °C at 50 μm) and it can stop a crystal from growing when held at a constant temperature within the thermal hysteresis gap. EfcIBP protects purified proteins and bacterial cells from freezing damage when exposed to challenging temperatures. EfcIBP also possesses a potential N-terminal signal sequence for protein transport and a DUF3494 domain that is common to secreted IBPs. These features lead us to hypothesize that the protein is either anchored at the outer cell surface or concentrated around cells to provide survival advantage to the whole cell consortium.

Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation

ABSTRACTTo survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.

A Supramolecular Ice Growth Inhibitor.

Safranine O, a synthetic dye, was found to inhibit growth of ice at millimolar concentrations with an activity comparable to that of highly evolved antifreeze glycoproteins. Safranine inhibits growth of ice crystals along the crystallographic a-axis, resulting in bipyramidal needles extended along the <0001> directions as well as and plane-specific thermal hysteresis (TH) activity. The interaction of safranine with ice is reversible, distinct from the previously reported behavior of antifreeze proteins. Spectroscopy and molecular dynamics indicate that safranine forms aggregates in aqueous solution at micromolar concentrations. Metadynamics simulations and aggregation theory suggested that as many as 30 safranine molecules were preorganized in stacks at the concentrations where ice growth inhibition was observed. The simulations and single-crystal X-ray structure of safranine revealed regularly spaced amino and methyl substituents in the aggregates, akin to the ice-binding site of antifreeze proteins. Collectively, these observations suggest an unusual link between supramolecular assemblies of small molecules and functional proteins.

A Simple and Quantitative Method to Evaluate Ice Recrystallization Kinetics Using the Circle Hough Transform Algorithm

The formation of large ice crystals via recrystallization processes in foods and water-based materials often decreases the quality and structural integrity of the materials. Hence, there is a widespread academic and commercial interest in natural and synthetic ice crystal growth modifiers that inhibit the recrystallization of ice. Well-known natural ice crystal growth modifiers are antifreeze proteins (AFPs), which inhibit ice recrystallization by adsorbing on the surface of ice crystals. Reliable quantification of the ice recrystallization inhibition (IRI) efficiency is a long-sought goal. In this work, we describe a simple method to quantitatively evaluate IRI efficiency, based on automated image analysis using the circle Hough transform (CHT) algorithm. It enables robust and high throughput analysis of natural and synthetic ice recrystallization inhibitors. Here we use the method to evaluate the impact of a single-point mutation in the ice-binding site of QAE on its IRI activity. We find that the T18N mutant of QAE has virtually the same effective ice recrystallization inhibitory concentration as the wild-type QAE. This is in contrast to thermal hysteresis activity, evaluated by cryoscopy or sonocrystallization, where the mutation greatly decreases the activity.

Purification and Identification of Antifreeze Protein From Cold-Acclimated Oat (Avena sativa L.) and the Cryoprotective Activities in Ice Cream

Antifreeze proteins (AFPs) were extracted from cold-acclimated oat by vacuum infiltration–centrifugation. Ammonium precipitation, ion exchange, and size-exclusion chromatography were used successively to purify the oat AFPs (AsAFPs), and the proteins were identified using matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry. The results showed that an ammonium-sulfate saturation of 50–100 % was optimal for supernatant precipitation. After purification, AsAFP was found to have achieved an electrophoretic purity and its thermal-hysteresis activity was measured at 1.24 °C (15 mg ml−1). The mass fingerprinting and sequencing results indicated the homology of AsAFP and endochitinase. Recrystallization and melting resistance of ice cream were improved by AsAFP (0.1 %). Moreover, the addition of AsAFP (0.1 %) in ice cream increased the glass transition temperature (Tg) from −29.14 to −27.74 °C.

Ice-Active Substances from the Infective Juveniles of the Freeze Tolerant Entomopathogenic Nematode, Steinernema feltiae.

Steinernema feltiae is a moderately freezing tolerant nematode, that can withstand intracellular ice formation. We investigated recrystallization inhibition, thermal hysteresis and ice nucleation activities in the infective juveniles of S. feltiae. Both the splat cooling assay and optical recrystallometry indicate the presence of ice active substances that inhibit recrystallization in the nematode extract. The substance is relatively heat stable and largely retains the recrystallization inhibition activity after heating. No thermal hysteresis activity was detected but the extract had a typical hexagonal crystal shape when grown from a single seed crystal and weak ice nucleation activity. An ice active substance is present in a low concentration, which may be involved in the freezing survival of this species by inhibiting ice recrystallization.

New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect.

The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at temperatures below -30°C. To investigate the deep supercooling ability of E. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins (HSPs) were identified. However, no antifreeze proteins (AFPs) were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97°C, suggesting its potential role in the deep supercooling ability of E. pela.

Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of commonly used colligative antifreezes such as salts and alcohols, the advantage of using AFPs as an additive is that they do not alter the physicochemical properties of the water-based material. Here, we report the first comprehensive evaluation of thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity of all major classes of AFPs using cryoscopy, sonocrystallization, and recrystallization assays. The results show that TH activities determined by cryoscopy and sonocrystallization differ markedly, and that TH and IRI activities are not correlated. The absence of a distinct correlation in antifreeze activity points to a mechanistic difference in ice growth inhibition by the different classes of AFPs: blocking fast ice growth requires rapid nonbasal plane adsorption, whereas basal plane adsorption is only relevant at long annealing times and at small undercooling. These findings clearly demonstrate that biomimetic analogs of antifreeze (glyco)proteins should be tailored to the specific requirements of the targeted application.

저온 유도 시스템을 가진 재조합 대장균을 이용한 남극 세균 Flavobacterium frigoris PS1 유래 결빙방지단백질의 Pilot-scale 생산

Antifreeze proteins (AFP) inhibit growth and recrystallization of ice, and permit organisms to survive in cold environments. The AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced in E. coli using a cold shock induction system. The culture temperature was shifted from <TEX>$37^{\circ}C$</TEX> to <TEX>$15^{\circ}C$</TEX> and a 20 L culture scale was used. The final weights of dried cell and FfIBP were estimated to be 126 g and 8.4 g, respectively. The thermal hysteresis (TH) activity (<TEX>$1.53^{\circ}C$</TEX>) of the produced FfIBP was 3.6-fold higher than that of the LeIBP (Leucosporidium ice-binding protein) produced in Picha. The current study demonstrates that large-scale production of FfIBP was successful and the result could be extended to further application studies using recombinant AFPs.

21. A lesson from Nature for controlling ice during organ cryopreservation by vitrification

Banking of organs using current tissue banking practices employing conventional cryopreservation by freezing is not feasible due to the well known damage caused by intra and extra-cellular ice formation. An alternative cryopreservation approach is known as vitrification. There has been considerable success in ice-free vitrification of small tissue samples but better ice control is required for samples > 10 mL in volume. Development of novel cryoprotectants and methods that will facilitate clinically effective banking by vitrification of large vascularized tissues and organs is needed. There are lessons to be learned for organ vitrification from Nature. Freeze avoiding insects such as Cujucus clavipes beetle larvae from the interior of Alaska vitrify and survive temperatures that routinely reach −60 °C with some individuals surviving exposure to −100 °C. At the onset of winter these insects combine changes in behavior with increases in cryoprotectant content (such as glycerol, proline, trehalose), dehydration, production of antifreeze compounds (proteins and glycolipids), and diapause. The cryoprotectant concentration can achieve 4.2 M, approximately half the concentration required for ice-free cryopreservation of tissue and organ therapy products. Recombinant copies of insect-derived antifreeze proteins are available, so they can be manufactured, and they are up to 10 times more effective in ice modulation (thermal hysteresis) than the well known fish-derived antifreeze proteins that have proven ineffective or detrimental for cell cryopreservation. Recently we have discovered that certain insect-derived antifreeze proteins significantly increase cell viability during cryopreservation by freezing. Similarly, in unpublished studies we have found that an insect-derived antifreeze glycolipid, with thermal hysteresis activity similar to that of hyperactive insect antifreeze proteins, can significantly increase cell viability post-cryopreservation using freezing methods. These glycolipids have been found in several species including freeze tolerant and avoiding insects, freeze tolerant frogs, and freeze tolerant plants. Unlike the antifreeze proteins, which are diverse in structure across species, these glycolipids are nearly identical. While we anticipate that eventually it will be possible to synthesize the saccharide component (s1-4 linked xylose-mannose), at present the antifreeze glycolipid is harvested from natural sources, especially plants. Translation of this lesson from Nature requires a better understanding of how animals prepare themselves for diapause and hibernation so that we can reproduce these phenomena in organ donors in vivo, under physiological conditions post-mortem prior to organ procurement, and/or organs ex-vivo before cryopreservation in cryoprotectant formulations that incorporate antifreeze compounds. It is anticipated that utilization of antifreeze proteins and glycolipids will enable utilization of less cytotoxic cryoprotectant formulations with enhanced ice control during cooling and warming of larger biological structures than currently possible. In the big picture it is likely that they will be combined with other strategies presented in this session on controlling ice formation including improved cryoprotectant addition and removal methods, pressure assisted cooling, stress relief by annealing, synthetic antifreeze compounds, and nanoheater assisted rewarming. Once we have ice formation under control we must be concerned with ischemic reperfusion injury, the final hurdle upon in vivo transplantation in patients.

Animal ice-binding (antifreeze) proteins and glycolipids: an overview with emphasis on physiological function.

Ice-binding proteins (IBPs) assist in subzero tolerance of multiple cold-tolerant organisms: animals, plants, fungi, bacteria etc. IBPs include: (1) antifreeze proteins (AFPs) with high thermal hysteresis antifreeze activity; (2) low thermal hysteresis IBPs; and (3) ice-nucleating proteins (INPs). Several structurally different IBPs have evolved, even within related taxa. Proteins that produce thermal hysteresis inhibit freezing by a non-colligative mechanism, whereby they adsorb onto ice crystals or ice-nucleating surfaces and prevent further growth. This lowers the so-called hysteretic freezing point below the normal equilibrium freezing/melting point, producing a difference between the two, termed thermal hysteresis. True AFPs with high thermal hysteresis are found in freeze-avoiding animals (those that must prevent freezing, as they die if frozen) especially marine fish, insects and other terrestrial arthropods where they function to prevent freezing at temperatures below those commonly experienced by the organism. Low thermal hysteresis IBPs are found in freeze-tolerant organisms (those able to survive extracellular freezing), and function to inhibit recrystallization - a potentially damaging process whereby larger ice crystals grow at the expense of smaller ones - and in some cases, prevent lethal propagation of extracellular ice into the cytoplasm. Ice-nucleator proteins inhibit supercooling and induce freezing in the extracellular fluid at high subzero temperatures in many freeze-tolerant species, thereby allowing them to control the location and temperature of ice nucleation, and the rate of ice growth. Numerous nuances to these functions have evolved. Antifreeze glycolipids with significant thermal hysteresis activity were recently identified in insects, frogs and plants.

Influence of antifreeze proteins on the ice/water interface.

Antifreeze proteins (AFP) are responsible for the survival of several species, ranging from bacteria to fish, that encounter subzero temperatures in their living environment. AFPs have been divided into two main families, moderately and hyperactive, depending on their thermal hysteresis activity. We have studied one protein from both families, the AFP from the snow flea (sfAFP) and from the winter flounder (wfAFP), which belong to the hyperactive and moderately active family, respectively. On the basis of molecular dynamics simulations, we have estimated the thickness of the water/ice interface for systems both with and without the AFPs attached onto the ice surface. The calculation of the diffusion profiles along the simulation box allowed us to measure the interface width for different ice planes. The obtained widths clearly show a different influence of the two AFPs on the ice/water interface. The different impact of the AFPs here studied on the interface thickness can be related to two AFPs properties: the protein hydrophobic surface and the number of hydrogen bonds that the two AFPs faces form with water molecules.

Modulation of antifreeze activity and the effect upon post-thaw HepG2 cell viability after cryopreservation

Most antifreeze proteins (AFPs) exhibit two types of “antifreeze activity” – thermal hysteresis (TH) and ice recrystallization inhibition (IRI) activity. The mechanism of TH activity has been studied in depth and is the result of an adsorption of AFPs to the surface of ice with an ice-binding face (IBF). In contrast, the mechanism of ice recrystallization and its inhibition is considerably less understood. In this paper, we examine several different antifreeze proteins, glycoproteins and mutants of the Lolium perenne AFP (LpAFP) to understand how IRI activity is modulated independently of TH activity. This study also examines the ability of the various AF(G)Ps to protect HepG2 cells from cryoinjury. Post-thaw cell viabilities are correlated to TH, IRI activity as well as dynamic ice shaping ability and single ice crystal growth progressions. While these results demonstrate that AF(G)Ps are ineffective as cryoprotectants, they emphasize how ice crystal habit and most importantly, ice growth progression affect HepG2 cell survival during cryopreservation.