Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.
Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.
Lin
Q
Q
Division of Structural Biology and Biochemistry, Hospital for Sick Children and the Department of Laboratory Medicine & Pathobiology, University of Toronto, Ontario, Canada.
Ewart
K V
KV
Yan
Q
Q
Wong
W K
WK
Yang
D S
DS
Hew
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D002942:Circular Dichroism; D005432:Flounder; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D012867:Skin / Q000378:metabolism
D021301:Antifreeze Proteins; D006023:Glycoproteins