title: Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.
abstract: The approximately 24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop beta-helix containing the approximately 24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each beta-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.
authors.affiliation: The Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Dang-Quan
authors.initials: DQ
authors.lastname: Zhang
authors.forename: Bing
authors.initials: B
authors.lastname: Liu
authors.forename: Dong-Ru
authors.initials: DR
authors.lastname: Feng
authors.forename: Yan-Ming
authors.initials: YM
authors.lastname: He
authors.forename: Shu-Qi
authors.initials: SQ
authors.lastname: Wang
authors.forename: Hong-Bin
authors.initials: HB
authors.lastname: Wang
authors.forename: Jin-Fa
authors.initials: JF
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence / Q000502:physiology; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D001216:Asparagine / Q000502:physiology*; D017124:Conserved Sequence / Q000502:physiology; D018552:Daucus carota / Q000737:chemistry; D005615:Freezing; D007053:Ice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000502:physiology
chemical_list: C114788:AFP protein, Daucus carota; D021301:Antifreeze Proteins; D007053:Ice; D010940:Plant Proteins; D001216:Asparagine