Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.
The survival strategies of polar organisms at permanently or extremely cold temperatures and their application to cryobiology were reviewed here. In addition, ongoing studies on psychrophiles also were described. Psychrophiles are extremophiles that can grow and reproduce in cold temperatures, typically at -10 to $20^{\circ}C$. These organisms developed various mechanisms of adaptation to extremely cold environments. Polar organisms cope with these extreme physicochemical conditions using strategies such as avoidance, protection and partnership with other organisms. Understanding on the strategies adopted by polar organisms may provide insight on the physiological process that cells can go through during freezing. Cryopreservation may be able to take advantage of the findings described above. Currently, genomes of many cold-loving organisms have been sequenced and comparative genomics has revealed, at a molecular level, the characteristics of these organisms. The investigation of microorganisms on the polar glaciers may expand our understanding on the origin of life on Earth and other planets.
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