Carrot ‘antifreeze’ protein has an irregular ice-binding site that confers weak freezing point depression but strong inhibition of ice recrystallization

摘要

Ice-binding proteins (IBPs) are found in many biological kingdoms where they protectorganisms from freezing damage as antifreeze agents or inhibitors of ice recrystallization.Here, the crystal structure of recombinant IBP from carrot (Daucus carota) has beensolved to a resolution of 2.3 ?. As predicted, the protein is a structural homologue of aplant polygalacturonase-inhibiting protein forming a curved solenoid structure with aleucine-rich repeat motif. Unexpectedly, close examination of its surface did not revealany large regions of flat, regularly spaced hydrophobic residues that characterize the ice-binding sites (IBSs) of potent antifreeze proteins from freeze-resistant fish and insects. AnIBS was defined by site-directed mutagenesis of residues on the convex surface of thecarrot solenoid. This imperfect site is reminiscent of the irregular IBS of grass ‘antifreeze’protein. Like the grass protein, the carrot IBP has weak freezing point depression activitybut is extremely active at nanomolar concentrations in inhibiting ice recrystallization. Icecrystals formed in the presence of both plant proteins grow slowly and evenly in all direc-tions. We suggest that this slow, controlled ice growth is desirable for freeze tolerance.The fact that two plant IBPs have evolved very different protein structures to affect ice ina similar manner suggests this pattern of weak freezing point depression and strong icerecrystallization inhibition helps their host to tolerate freezing rather than to resist it.