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Hipposin is a potent 51-mer antimicrobial peptide (AMP) from Atlantic halibut with sequence similarity to parasin (19-mer catfish AMP), buforin I (39-mer toad AMP), and buforin II (an active 21-mer fragment of buforin I), suggesting that the antimicrobial activity of these peptides might all be due to a common antimicrobial sequence motif. In order to identify the putative sequence motif, the antimicrobial activity of hipposin fragments against 20 different bacteria was compared to the activity of hipposin, parasin and buforin II. Neither parasin nor the 19-mer parasin-like fragment HIP(1-19) (differs from parasin in only three residues) that is derived from the N-terminal part (residues 1-19) of hipposin had marked antimicrobial activity. In contrast, the fragment HIP(16-36) (identical to buforin II) that is derived from the middle part of hipposin (residues 16-36) had such activity, indicating that this part of hipposin contained an antimicrobial sequence motif. The activity was enhanced when the parasin-like N-terminal sequence was also present, as the fragment HIP(1-36) which consists of residues 1-36 in hipposin was more potent than HIP(16-36). Extending HIP(1-36) with three C-terminal residues-thereby constructing the buforin I-like peptide HIP(1-39) (differs from buforin I in only three residues)-increased the activity further. Also, the presence of the C-terminal part of hipposin (residues 40-51) increased the activity, as hipposin was clearly the most potent of all the peptides that were tested. Circular dichroism structural analysis of the peptides revealed that they were all non-structured in aqueous solution. However, trifluoroethanol and the membrane-mimicking entities dodecylphosphocholine micelles and negatively charged liposomes induced (amphiphilic) alpha-helical structuring in hipposin. Judging from the structuring of the individual fragments, the tendency for alpha-helical structuring appeared to be greater in the C-terminal and the buforin II-like middle region of hipposin than in the parasin-like N-terminal region.