Figure 9. Predicted membrane binding modes (left) and lipophilicity profiles (right) for kalata B1 (A), cycloviolacin O2 (B) and kalata B8 (C) in their native cyclic conformations and in hypothetically unfolded conformations.

The outer phosphate layer of a phospholipid membrane is represented by a red line in each case. Strained backbone regions with little structural mobility are indicated in blue. Backbone regions with a defined secondary structure are colored in yellow and red to indicate β-strands and α-helices, respectively. Only selected side chains are depicted, including those that form the hydrophobic patch, residues carrying positive charges, and the conserved glutamic acid in loop 1. The cyclotide structures were obtained from the PDB server; their PDB IDs are 1NB1 (kalata B1) [28], 2KNM (cycloviolacin O2) [33], and 2B38 (kalata B8) [16]. The lipophilicity profile of each residue was calculated for the natively folded cyclotides and for their unfolded conformations. The hypothetical unfolded cyclotide conformations were defined such that ψ = φ = 180°. It should be noted that in the natively folded conformations of kalata B1 (kB1) and cycloviolacin O2 (cyO2), the degree of solvent exposure for lipophilic residues is high while that for lipophobic residues is low. For example, in loop 2 of cyO2, the lipophilicity of Trp and Pro is higher in the native fold than in the unfolded conformation. Moreover, the lipophobicity values for polar residues are relatively high in natively folded kB8 compared to natively folded kB1 and cyO2. In contrast to the situation for kB1 and cyO2, the polar residues of kB8 exhibit similar lipophobicities in the native and unfolded conformations. Notably, the glutamic acid residue in loop 1 of kB8 participates in an internal hydrogen-bonding network within the core of the protein that effectively minimizes its SASA value. Consequently, it exhibits a much higher level of lipophobicity in the native fold than do the equivalent residues of kB1 and cyO2.