Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.
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.
Zhang
Dang-Quan
DQ
The Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
Liu
Bing
B
Feng
Dong-Ru
DR
He
Yan-Ming
YM
Wang
Shu-Qi
SQ
Wang
Hong-Bin
HB
Wang
Jin-Fa
JF
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
C114788:AFP protein, Daucus carota; D021301:Antifreeze Proteins; D007053:Ice; D010940:Plant Proteins; D001216:Asparagine