Inhibition of cryoaggregation of phospholipid liposomes by an Arabidopsis intrinsically disordered dehydrin and its K-segment.

Dehydrin is an intrinsically disordered protein involved in the cold tolerance of plants. Although dehydrins have been thought to protect biomembranes under cold conditions, the underlying protective mechanism has not been confirmed. Here we report that Arabidopsis dehydrin AtHIRD11 inhibited the aggregation of phospholipid liposomes after freezing and thawing. AtHIRD11 showed significantly greater cryoaggregation-prevention activity than cryoprotective agents such as trehalose, proline, and polyethylene glycols. Amino acid sequence segmentation analysis indicated that the K-segment of AtHIRD11 inhibited the cryoaggregation of phosphatidylcholine (PC) liposomes but other segments did not. This showed that K-segments conserved in all dehydrins were likely to be the cryoprotective sites of dehydrins. Amino acid replacement for a typical K-segment (TypK for short) sequence demonstrated that both hydrophobic and charged amino acids were required for the cryoaggregation-prevention activity of PC liposomes. The amino acid shuffling of TypK remarkably reduced cryoprotective activity. Although TypK did not bind to PC liposomes in solution, the addition of liposomes reduced its disordered content under crowded conditions. Together, these results suggested that dehydrins protected biomembranes via conserved K-segments whose sequences were optimized for cryoprotective activities.

Cryoprotective activity of Arabidopsis KS-type dehydrin depends on the hydrophobic amino acids of two active segments.

Dehydrins are intrinsically disordered proteins which are related to cold tolerance in plants. Dehydrins show potent cryoprotective activities for freeze-sensitive enzymes such as lactate dehydrogenase (LDH). Previous studies demonstrated that K-segments conserved in dehydrins had cryoprotective activities and that K-segment activities depended on the hydrophobic amino acids in the segment. However, the cryoprotective roles of hydrophobic amino acids in dehydrin itself have not been reported. Here, we demonstrated that hydrophobic amino acids were required for the cryoprotective activity of Arabidopsis dehydrin AtHIRD11. Cryoprotective activities were compared between AtHIRD11 and the corresponding mutant in which all hydrophobic residues were changed to T (AtHIRD11Φ/T) by using LDH. The change strikingly reduced AtHIRD11 activity. A segmentation analysis indicated that the conserved K-segment (Kseg) and a previously unidentified segment (non-K-segment 1, NK1) showed cryoprotective activities. Circular dichroism indicated that the secondary structures of all peptides showed disorder, but only cryoprotective peptides changed to the ordered forms by sodium dodecyl sulfate. Ultracentrifuge analysis indicated that AtHIRD11 and AtHIRD11Φ/T had similar molecular sizes in solution. These results suggest that not only structural disorder but also hydrophobic amino acids contributed to the cryoprotective activity of AtHIRD11. A possible mechanism based on an extended molecular shield model is proposed.

F-segments of Arabidopsis dehydrins show cryoprotective activities for lactate dehydrogenase depending on the hydrophobic residues.

Although dehydrins show cryoprotective activities for freeze-sensitive enzymes, the underlying mechanism is still under investigation. Here, we report that F-segments conserved in some dehydrins cryoprotected lactate dehydrogenase (LDH) as well as K-segments, which were previously identified as cryoprotective segments of dehydrins. The cryoprotective activity levels of four F-segments of Arabidopsis dehydrins were similar to that of a typical K-segment. Amino acid substitution experiments indicated that the activity of the F-segment of Arabidopsis COR47 (designated as Fseg) depended on the hydrophobic residues (L, F, and V). Intriguingly, when all the amino acids other than the hydrophobic residues were changed to glycine, the cryoprotective activity did not change, suggesting that the hydrophobic amino acids were sufficient for Fseg activity. Circular dichroism analysis indicated that Fseg was mainly disordered in aqueous solution as well as Fseg_Φ/T, in which the hydrophobic residues of Fseg were changed to T. This suggested that the hydrophobic interaction might be related to the cryoprotective activities of Fseg.