New Cysteine-Rich Ice-Binding Protein Secreted from Antarctic Microalga, Chloromonas sp.
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.
Department of Life Science, Hanyang University, Seoul, South Korea.
Woongsic
W
Jung
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada K7L-3N6.
Robert L
http://orcid.org/0000-0002-5473-5876
RL
Campbell
Department of Life Science, Hanyang University, Seoul, South Korea.
Yunho
Y
Gwak
Department of Biology, Chungnam National University, Daejeon, South Korea.
Jong Im
JI
Kim
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Canada K7L-3N6.
Peter L
PL
Davies
Department of Life Science, Hanyang University, Seoul, South Korea.
EonSeon
E
Jin
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D000460:Chlorophyta / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007053:Ice / Q000032:analysis*; D058086:Microalgae / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D015854:Up-Regulation
D021301:Antifreeze Proteins; D007053:Ice; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D003545:Cysteine