The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins    

Abstract

Dehydrins protect plant proteins and membranes from damage during drought and cold. Vitis riparia K 2 is a 48-residue protein that can protect lactate dehydrogenase from freeze-thaw damage by preventing the aggregation and denaturation of the enzyme. To further elucidate its mechanism, we used a series of V. riparia K 2 concatemers (K 4 , K 6 , K 8 , and K 10 ) and natural dehydrins (V. riparia YSK 2 , 60 kilodalton peach dehydrin [PCA60], barley dehydrin5 [Dhn5], Thellungiella salsuginea dehydrin2 , and Opuntia streptacantha dehydrin1 ) to test the effect of the number of K-segments and dehydrin size on their ability to protect lactate dehydrogenase from freeze-thaw damage. The results show that the larger the hydrodynamic radius of the dehydrin, the more effective the cryoprotection. A similar trend is observed with polyethylene glycol, which would suggest that the protection is simply a nonspecific volume exclusion effect that can be manifested by any protein. However, structured proteins of a similar range of sizes did not show the same pattern and level of cryoprotection. Our results suggest that with respect to enzyme protection, dehydrins function primarily as molecular shields and that their intrinsic disorder is required for them to be an effective cryoprotectant. Lastly, we show that the cryoprotection by a dehydrin is not due to any antifreeze proteinlike activity, as has been reported previously.

Structural and Functional Insights into the Cryoprotection of Membranes by the Intrinsically Disordered Dehydrins

“…1B). Lysozyme was chosen as a control because it is a small protein with approximately the same hydrodynamic radius as K 2 (23) and is also known to bind to liposomes and promote their aggregation (44). As with dehydrins, lysozyme has previously been shown to bind to negatively charged liposomes (45).…”

“…The protein was lyophilized to dryness and stored at Ϫ20°C until use. K peptide was synthesized and purified as described previously (23).…”

“…The repeated freezing and thawing of lactate dehydrogenase results in its loss of activity, most likely because of aggregation (22). The addition of dehydrin results in the recovery of activity, with the longer the dehydrin the more efficient the recovery (23). Dehydrins have been shown to bind metals, which may prevent the formation of reactive oxygen species.…”

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure

“…Unlike other intrinsically disordered cold-stress proteins (e.g. Hughes et al, 2013), recombinant FST did not prevent the loss of LDH activity during freeze-thaw cycles, suggesting the protein has no role in protection of enzymes from freezing damage. We were unable to assess whether FST can protect against cold-induced (rather than freezinginduced) protein damage because of the lack of an established colddenaturation enzyme assay, so we cannot rule out the possibility that FST may function at more moderate low temperatures (e.g.…”

“…3B). We examined whether FST, like dehydrin, another disordered stress protein (Hughes et al, 2013), could protect LDH from damage caused by freezing and thawing the enzyme (Fig. 4).…”