title: Characterization of the ice-binding protein from Arctic yeast Leucosporidium sp. AY30.
abstract: Previously, we reported the ice-binding protein (LeIBP) from the Arctic yeast Leucosporidium sp. AY30. In this study we provide physicochemical characterization of this IBP, which belongs to a class of IBPs that exhibited no significant similarity in primary structure to other known antifreeze proteins (AFPs). We compared native, glycosylated and non-glycosylated recombinant LeIBPs. Interestingly, size-exclusion chromatography and analytical ultracentrifugation revealed that LeIBP self-associates with a reversible dimer with K(d) values in the range 3.45-7.24×10(-6) M. Circular dichroism (CD) spectra showed that LeIBP, glycosylated or non-glycosylated, is predominantly composed of β-strand secondary structural elements (54.6%), similar to other β-helical antifreeze proteins (AFPs). In thermal hysteresis (TH) activity measurements, native LeIBP was twice more active (0.87 °C at 15 mg/mL) than that of the recombinant IBPs (0.43-0.42 °C at 10.8 mg/mL). This discrepancy is probably due to uncharacterized enhancing factors carried over during ice affinity purification, because glycosylated and non-glycosylated recombinant proteins displayed similarly low activity. Ice recrystallization inhibition (RI) activities of the native and recombinant LeIBPs were comparable. Measurements of CD, TH activity, and RI showed that glycosylation does not cause structural changes and is not required for function. An ice-etching experiment using green fluorescent protein-tagged IBP revealed that LeIBP binds, just as hyperactive AFPs, to both basal and pyramidal prism planes of the ice crystal. Taken together, our results indicate that LeIBP, structurally similar to hyperactive AFPs, is moderately active and that a reversible dimer has no effect on its activity.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Kyoung Sun
authors.initials: KS
authors.lastname: Park
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.forename: Seung Il
authors.initials: SI
authors.lastname: Park
authors.forename: Eun jung
authors.initials: Ej
authors.lastname: Kim
authors.forename: Soon-Jong
authors.initials: SJ
authors.lastname: Kim
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Kang
authors.forename: Hak Jun
authors.initials: HJ
authors.lastname: Kim
mesh_terms: D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D001110:Arctic Regions; D001487:Basidiomycota / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D019281:Dimerization; D004926:Escherichia coli; D005615:Freezing; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D007053:Ice / Q000032:analysis*; D007700:Kinetics; D008958:Models, Molecular; D010843:Pichia; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
chemical_list: D021301:Antifreeze Proteins; D005656:Fungal Proteins; D007053:Ice; D011993:Recombinant Fusion Proteins