Lin Q, Ewart K V, Yan Q, Wong W K, Yang D S, Hew C L
Division of Structural Biology and Biochemistry, Hospital for Sick Children and the Department of Laboratory Medicine & Pathobiology, University of Toronto, Ontario, Canada.
Eur J Biochem. 1999 Aug;264(1):49-54. doi: 10.1046/j.1432-1327.1999.00569.x.
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.
