Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers

Graham, Ben; Bailey, Trisha L.; Healey, Joseph R. J.; Marcellini, Moreno; Deville, Sylvain; Gibson, Matthew I.

doi:10.1002/anie.201706703

Cited by 96 publications

(120 citation statements)

References 41 publications

(47 reference statements)

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“… 6 , 7 Gibson and co-workers have developed synthetic polymers 8 , 9 based upon poly(vinyl alcohol) and poly(ampholytes) which have been found to enhance the cryopreservation of blood 10 − 12 and nucleated cells. 4 , 13 …”

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confidence: 99%

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

et al. 2018

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Antifreeze glycoproteins (AFGPs) from polar fish are the most potent ice recrystallization (growth) inhibitors known, and synthetic mimics are required for low-temperature applications such as cell cryopreservation. Here we introduce facially amphipathic glycopolymers that mimic the three-dimensional structure of AFGPs. Glycopolymers featuring segregated hydrophilic and hydrophobic faces were prepared by ring-opening metathesis polymerization, and their rigid conformation was confirmed by small-angle neutron scattering. Ice recrystallization inhibition (IRI) activity was reduced when a hydrophilic oxo-ether was installed on the glycan-opposing face, but significant activity was restored by incorporating a hydrophobic dimethylfulvene residue. This biomimetic strategy demonstrates that segregated domains of distinct hydrophilicity/hydrophobicity are a crucial motif to introduce IRI activity, which increases our understanding of the complex ice crystal inhibition processes.

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confidence: 99%

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

et al. 2018

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“…Indeed, study of various length polyproline showed that it has ice recrystallization inhibition activity owing to its amphipathic PPII helix structure. 33 Therefore, the identification of PPII extended helical conformations for LEA and other similar peptides and proteins that exist in low hydration, high salinity, or extreme temperatures suggests the structure assists in damage prevention during desiccation, ionic and osmotic stress, and freezing by retaining water molecules. [34][35][36][37][38][39][40] For example, Soulages et al previously reported recombinant Group 1 LEA (GmD-19) and Group 2 LEA (GmDHN1) proteins from soybean existing in the extended PPII helical conformation in aqueous conditions and low temperatures.…”

Section: Discussionmentioning

confidence: 99%

The effect of salt and temperature on the conformational changes of P1LEA‐22, a repeat unit of plant Late Embryogenesis Abundant proteins

León

Vermeuel

Gupta

et al. 2020

Journal of Peptide Science

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The effect of choline chloride on the conformational dynamics of the 11‐mer repeat unit P1LEA‐22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA‐22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl3 or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α‐helix and β‐sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl3 and choline chloride salts on P1LEA‐22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage‐preventing measure of Late Embryogenesis Abundant proteins.

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“…Therefore, synthetic macromolecular mimics of AFPs have emerged, which can reproduce their properties but benefit from the scalable and tunable synthesis of synthetic polymers. 18,21,22 These have been used to enhance the cryopreservation of mammalian cells including blood, 23−25 cell lines, 26 and primary cells. 27,28 Their primary function is ice recrystallization inhibition, where the rate of growth (not formation) of ice is slowed, leading to reduced cell death during thawing.…”

Section: Introductionmentioning

confidence: 99%

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms

2018

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All modern molecular biology and microbiology is underpinned by not only the tools to handle and manipulate microorganisms but also those to store, bank, and transport them. Glycerol is the current gold-standard cryoprotectant, but it is intrinsically toxic to most microorganisms: only a fraction of cells survive freezing and the presence of glycerol can impact downstream applications and assays. Extremophile organisms survive repeated freeze/thaw cycles by producing antifreeze proteins which are potent ice recrystallization inhibitors. Here we introduce a new concept for the storage/transport of microorganisms by using ice recrystallization inhibiting poly(vinyl alcohol) in tandem with poly(ethylene glycol). This cryopreserving formulation is shown to result in a 4-fold increase in E. coli yield post-thaw, compared to glycerol, utilizing lower concentrations, and successful cryopreservation shown as low as 1.1 wt % of additive. The mechanism of protection is demonstrated to be linked not only to inhibiting ice recrystallization (by comparison to a recombinant antifreeze protein) but also to the significantly lower toxicity of the polymers compared to glycerol. Optimized formulations are presented and shown to be broadly applicable to the cryopreservation of a panel of Gram-negative, Gram-positive, and mycobacteria strains. This represents a step-change in how microorganisms will be stored by the design of new macromolecular ice growth inhibitors; it should enable a transition from traditional solvent-based to macromolecular microbiology storage methods.

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Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers

Cited by 96 publications

References 41 publications

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

Facially Amphipathic Glycopolymers Inhibit Ice Recrystallization

The effect of salt and temperature on the conformational changes of P1LEA‐22, a repeat unit of plant Late Embryogenesis Abundant proteins

Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms

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