Isolation and Characterization of Novel Cyclotides from Viola hederaceae

Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 (vhl1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional H NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded -sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pKa of 3.0 for Glu 3 and 5.0 for Glu to be determined. Met was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 M.