Winter flounder contains two distinct anti-freeze protein isoforms, which are 
the liver-type extracellular anti-freeze proteins and the skin-type 
intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit 
lower anti-freeze activities than the liver-type isoforms and this might be due 
to their lacking complete ice-binding motifs. One of the skin-type anti-freeze 
proteins, skin-type anti-freeze protein-3, does contain putative overlapping 
ice-binding motifs with the sequences '-K-DT-' and '-DT-K-'. Synthetic 
anti-freezes containing 0-3 repeats of the '-DT-K-' motif were tested for 
stability and activity. Loss of the single '-DT-K-' of skin-type anti-freeze 
protein-3 increases the anti-freeze activity and increasing the number of motifs 
to two or three lowers the activity. The decrease in activity with an increasing 
frequency of the motif correlates with a decrease in the helical content of 
these peptides at 0 degrees C.