Figure 3. The sequences of the membrane-buried regions of selected cyclotides and their lipophilicity profiles.

The multiple sequence alignment shows all of the cyclotides for which 3D structures (based on solution-phase NMR studies) are available. These sequences were used as template candidates for homology modeling of the cyclotides of unknown structure. The cysteine of loop 1 was arbitrarily selected as the N-terminus for the multiple sequence alignments. The residues required for cyclization, i.e. the conserved C-terminal residue (Asn or Asp) and N-terminal residue (Gly), are positioned within loop 6. The membrane-buried residues are highlighted with a gray background. OPM was used to predict the orientation of the selected cyclotides on the membrane. Among the members of the Möbius subfamily, many of the loop 5 and 6 residues are membrane-buried. Among the bracelet cyclotides, large proportions of loops 2 and 3 are buried. Only a couple of residues from the cyclotides of the hybrid subfamily penetrate the membrane interface. The lipophilicity of the loops was calculated as the total lipophilicity (L_S) of all of the residues within the loop. It should be noted that the membrane-buried regions identified by OPM are consistent with those exhibiting high lipophilicity.