RATIONAL DESIGN AND SYNTHESIS OF ANTIFREEZE-PROTEIN INSPIRED POLYMERS FOR ANTI-ICING COATINGS AND CRYOPRESERVATION APPLICATIONS

Certain organisms living in cold regions have adapted different strategies to survive in harshly cold temperatures. Some of them use freeze-avoiding strategies in which they can prevent freezing by controlling the concentration of sugars (et. sucrose, trehalose) or polyols (glycerol), regulation of the ice nucleator, and dehydration. Other organisms have adapted to this extremely cold condition by producing antifreeze (gly)proteins (AF(G)Ps) which exhibit ice recrystallization inhibition (IRI), thermal hysteresis activity (THA), and dynamic ice crystal shaping. These proteins discovered in Antarctic fish in 1960 for the first time have been reported in bacteria, fungi, insects, and plants. AF(G)Ps and their synthetic biomimetics have received increasing attention as potential candidates for various industrial and bio-medical applications. Promising results from vitrification and other protocols using antifreeze agents with ice recrystallization inhibition activity have widely been reported in biopreservation. Conversely, understanding of the antifreezing process caused by these macromolecules remains under challenge. This is due to the multifunctional nature of the freezing process and antifreeze macromolecule’s behavior which brings complexity in designing the synthetic antifreeze structures. In addition, the cost, low availability, toxicity at higher concentrations, and instability beside several other drawbacks make their large-scale production challenging. Although several synthetic attempts for the exploitation of AFPs have been studied in the past, challenges remain in the synthetic design of AFP analogs. On the other hand, poly (vinyl alcohol) (PVA) with simple structure has been reported with potent IRI activity as a good candidate for large-scale production and applications. Our group has explored structural variations to polyol-based polymers to contrast with PVA as a control and identified several key structural elements for performance in IRI, THA, as well as in ice nucleation inhibition (INI). These structural features are bioinspired by the typical ice-binding plane of AFPs yet are surprisingly simple to produce with potency approaching that of typical AFPs. Key to the performance is positioning small organic functionalities with known antifreeze properties (glycerol) pendent to a host polymer chain with consideration of their conformational freedom. To build systematic variations into both the backbone and side-chain structures, we used poly (vinyl alcohol), poly (isopropenyl acetate), poly (acrylic acid), and poly(methacrylic acid) parent polymers for such pendent modifications. One structure in particular, glycerol-grafted-PVA (G-g-PVA), shows potency rivaling that of AFPs at similar micromolar concentration. The findings in this study help guide the rational design of synthetic antifreeze polymers useful for applications such as anti-icing coatings through to cryopreservation methods for organ transport and cell preservation. While AFPs are well-known for their ice nucleation and recrystallization inhibition activity along with controlling the ice crystal morphology, the contrasting behavior of ice nucleation promotion by AFPs and its key contribution to the whole antifreezing process also seems necessary to explore in this context. Here, silver iodide (AgI) has been used as an ice nucleator in different polymer solutions in ultra-pure water (UPW) to imitate the ice nucleation process by AFPs. PVA prepared by RAFT polymerization and our glycerol grafted derivative (G-g-PVA), now shown to be the most IRI active polymer to date, was investigated for its ice nucleation and recrystallization activity in AgI dispersion media. The results showed that the ice nucleation rate and temperature was significantly changed by adding the AgI dispersion in PVA and G-g-PVA solutions. The polymer solution in UPW containing AgI dispersion showed significant improvement in IRI activity compared the same polymer in PBS buffer solution. Our results demonstrate the considerable contribution of the ice nucleator in ice nucleation rate and temperature which enhances IRI activity of synthetic antifreeze polymers. These finding both aid our understanding of the ice nucleation promotion impact on synthetic polymers IRI activity along with engineering biomimetics for biomedical and industrial applications. Next, we focused our efforts to transfer these functionalities and performance to the solid-state interface with water. Aqueous dispersions of polymeric colloidal particles served as this substrate and were functionalized with either PVA or G-g-PVA grafted to their surfaces to contrast with performance of the same polymers strictly in the solution state. These functionalized colloids also can be applied as a continuous coating through latex film formation to assess anti-icing and ice-adhesion properties. While these systems also showed encouraging and potent activity, their performance was not enhanced compared to that of the solution state systems. This may have implications for fully solid-state anti-icing coatings, yet our attention then shifted from this scope of work to new funding which required again a solution state approach. In this final application, we explored PVA and G-g-PVA synthesized in our lab for their biopreservation aspects especially for red blood cell (RBC) cryopreservation at -80 °C. Our results again confirmed G-g-PVA to be an excellent candidate for cryopreservation and quite likely for organ cryopreservation. Using this polymer in solution as a cryoprotectant for RBCs showed significant improvement to controls, preventing hemolysis (cell rupture) along with eliminating other drawbacks that have been observed when using small molecule cryoprotective agents like glycerol, dimethyl sulfoxide (DMSO), etc.; especially with regard to the ability to fully remove all traces of the cryoprotectant after cryopreservation storage and thawing. In summary, the studies in this dissertation provide critical insights and approaches for the understanding of the freezing process and ideas that can help understand relevant mechanisms of influencing key freezing steps that have not yet been fully understood. In addition, it provides guidelines to synthesize G-g-PVA, currently the most potent active polymer in terms of IRI and THA shown useful for several high impact applications. In particular, this research provides valuable data and experimental conditions to understand the IRI mechanism to use in engineering next generation highly efficient antifreeze systems

In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts.

The spindle pole body (SPB) is the equivalent of the centrosome in fission yeast. In vivo it nucleates microtubules (MTs) during mitosis, but, unlike animal centrosomes, does not act as a microtubule organizing center (MTOC) during interphase. We have studied the MT-nucleating activity of SPBs in vitro and have found that SPBs in permeabilized cells retain in vivo characteristics. SPBs in cells permeabilized during mitosis can nucleate MTs, and are recognized by two antibodies: anti-gamma-tubulin and MPM-2 which recognizes phosphoepitopes. SPBs in cells permeabilized during interphase cannot nucleate MTs and are only recognized by anti-gamma-tubulin. Interphase SPBs which cannot nucleate can be converted to a nucleation competent state by incubation in cytostatic factor (CSF)-arrested Xenopus egg extracts. After incubation, they are recognized by MPM-2, and can nucleate MTs. The conversion does not occur in Xenopus interphase extract, but occurs in Xenopus interphase extract driven into mitosis by preincubation with exogenous cyclin B. The conversion is ATP dependent and inhibited by protein kinase inhibitors and alkaline phosphatase. Purified, active, cdc2 kinase/cyclin B complex in itself is not effective for activation of MT nucleation, although some interphase SPBs are now stained with MPM-2. These results suggest that the ability of SPBs in vitro to nucleate MTs after exposure to CSF-arrested extracts is activated through a downstream pathway which is regulated by cdc2 kinase

Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs

Bioinspired Multifunctional Anti-icing Hydrogel

The recent anti-icing strategies in the state of the art mainly focused on three aspects: inhibiting ice nucleation, preventing ice propagation, and decreasing ice adhesion strength. However, it is has proved difficult to prevent ice nucleation and propagation while decreasing adhesion simultaneously, due to their highly distinct, even contradictory design principles. In nature, anti-freeze proteins (AFPs) offer a prime example of multifunctional integrated anti-icing materials that excel in all three key aspects of the anti-icing process simultaneously by tuning the structures and dynamics of interfacial water. Here, inspired by biological AFPs, we successfully created a multifunctional anti-icing material based on polydimethylsiloxane-grafted polyelectrolyte hydrogel that can tackle all three aspects of the anti-icing process simultaneously. The simplicity, mechanical durability, and versatility of these smooth hydrogel surfaces make it a promising option for a wide range of anti-icing applications

Dynamical mechanism of antifreeze proteins to prevent ice growth

The fascinating ability of algae, insects and fishes to survive at temperatures below normal freezing is realized by antifreeze proteins (AFPs). These are surface-active molecules and interact with the diffusive water/ice interface thus preventing complete solidification. We propose a new dynamical mechanism on how these proteins inhibit the freezing of water. We apply a Ginzburg-Landau type approach to describe the phase separation in the two-component system (ice, AFP). The free energy density involves two fields: one for the ice phase with a low AFP concentration, and one for liquid water with a high AFP concentration. The time evolution of the ice reveals microstructures resulting from phase separation in the presence of AFPs. We observed a faster clustering of pre-ice structure connected to a locking of grain size by the action of AFP, which is an essentially dynamical process. The adsorption of additional water molecules is inhibited and the further growth of ice grains stopped. The interfacial energy between ice and water is lowered allowing the AFPs to form smaller critical ice nuclei. Similar to a hysteresis in magnetic materials we observe a thermodynamic hysteresis leading to a nonlinear density dependence of the freezing point depression in agreement with the experiments

A role for Gle1, a regulator of DEAD-box RNA helicases, at centrosomes and basal bodies.

Control of organellar assembly and function is critical to eukaryotic homeostasis and survival. Gle1 is a highly conserved regulator of RNA-dependent DEAD-box ATPase proteins, with critical roles in both mRNA export and translation. In addition to its well-defined interaction with nuclear pore complexes, here we find that Gle1 is enriched at the centrosome and basal body. Gle1 assembles into the toroid-shaped pericentriolar material around the mother centriole. Reduced Gle1 levels are correlated with decreased pericentrin localization at the centrosome and microtubule organization defects. Of importance, these alterations in centrosome integrity do not result from loss of mRNA export. Examination of the Kupffer's vesicle in Gle1-depleted zebrafish revealed compromised ciliary beating and developmental defects. We propose that Gle1 assembly into the pericentriolar material positions the DEAD-box protein regulator to function in localized mRNA metabolism required for proper centrosome function

X-ray diffraction to probe the kinetics of ice recrystallization inhibition

Understanding the nucleation and growth of ice is crucial in fields ranging from infrastructure maintenance, to the environment, and to preserving biologics in the cold chain. Ice binding and antifreeze proteins are potent ice recrystallization inhibitors (IRI), and synthetic materials that mimic this function have emerged, which may find use in biotechnology. To evaluate IRI activity, optical microscopy tools are typically used to monitor ice grain size either by end-point measurements or as a function of time. However, these methods provide 2-dimensional information and image analysis is required to extract the data. Here we explore using wide angle X-ray scattering (WAXS/X-ray powder diffraction (XRD)) to interrogate 100's of ice crystals in 3-dimensions as a function of time. Due to the random organization of the ice crystals in the frozen sample, the number of orientations measured by XRD is proportional to the number of ice crystals, which can be measured as a function of time. This method was used to evaluate the activity for a panel of known IRI active compounds, and shows strong agreement with results obtained from cryo-microscopy, as well as being advantageous in that time-dependent ice growth is easily extracted. Diffraction analysis also confirmed, by comparing the obtained diffraction patterns of both ice binding and non-binding additives, that the observed hexagonal ice diffraction patterns obtained cannot be used to determine which crystal faces are being bound. This method may help in the discovery of new IRI active materials as well as enabling kinetic analysis of ice growth