Abstract
The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41-47 A. Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.
Highlights
Cyclotides display a diverse range of biological activities, including anti-human immunodeficiency virus [5,6,7,8], neurotensin antagonism [9], hemolytic [10], antimicrobial [11], antifouling [12], and pesticidal activities [13,14,15,16,17,18,19]
These findings indicate that cyclotides interact with a wide range of membranes, with incorporation of the cyclotide structure into monolayers, and result in leakage of cellular contents from bilayers (HeLa cells), a functional study of cyclotide-membrane interactions is to date lacking
The results indicate that the diverse range of biological activities reported for cyclotides can be accounted for by membrane permeabilization associated with transmembrane pores with channel-like activity
Summary
Protein Purification—Native kalata B1 was isolated from the above ground parts of Oldenlandia affinis and purified as described previously [4]. The concentration of the peptide was determined spectrophotometrically using an extinction coefficient of 5875 MϪ1 cmϪ1 at 280 nm. Synthesis of Alanine Mutants—Alanine mutants of kalata B1 were synthesized using solid-phase methods as previously described [26]. The peptides were assembled using manual solid-phase peptide synthesis with t-butoxycarbonyl chemistry. Amino acids were added to the resin using 2-(1-Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
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