For decades peptides and proteins have been regarded as linear chains of amino acids. The only exception were cyclic peptide antibiotics like cyclosporins, synthesized by multifunctional enzymes and characterised by their high content of unusual amino acids.
In the last few years, however, several circular proteins - true gene products with their backbone cyclised through a peptide bond - have been discovered in microorganisms as well as plants and even a mammal. The representatives of this new type of polypeptides range in size from 12 to 80 amino acids and generally display well defined three dimensional structures in solution. Two groups of circular peptides not only contain the characteristic backbone modification, but also multiple disulfide bonds. In the Rhesus θ defensins, found in Rhesus macaque leukocytes, the three disulfide bonds are arranged like the rungs of a ladder. In the CCK (cyclic
cystine knot) family, the peptide backbone is cross-braced by three disulfide bonds arranged in a cystine knot motif, where two disulfide bonds and the backbone segments between them form a ring that is penetrated by the third disulfide bond. The CCK peptides consist of two subgroups: cyclotides, isolated from Rubiaceae and Violaceae plants and with around 40 highly homologous peptides the larges group amongst the circular peptides, and trypsin inhibitors found in bitter gourd, a member of the Cucurbitaceae family.
The aim of this study was to broaden the knowledge about cyclotides as well as to compare the two types of disulfide-rich circular peptides, i.e. knotted cyclotides and ladder-like θ defensins.
Screening of over 100 plant species expanded the list of cyclotide expressing plants considerably, although no cyclotides could be found in plants of families other than the Rubiaceae or
Violaceae. It was established that the expression of cyclotides not only varies with the plant species, but also with the tissue type (e.g. roots, leaves, flowers) of an individual plant as well as with various environmental factors. Four cyclotides were isolated from various Violaceae plants and further characterised.
One member of the Rhesus θ defensins, RTD-1, and its open chain analogue oRTD-1 were synthesized and their solution structures determined. In contrast to the cyclotides, which show very well defined, almost rigid structures, RTD-1 and oRTD-1 displayed considerable flexibility in the β-sheet part of the molecule, both in aqueous solution and in a micellar environment. Since cyclotides and Rhesus θ defensins are active against various microorganisms, their interactions with membrane mimics were investigated by NMR relaxation studies. None of the peptides tested (kalata B1, RTD-1 and oRTD-1) showed interaction with
sodium dodecyl sulfate micelles. With dihex-anoylphosphatidylcholine, however, all three peptides changed the internal mobility of the micelle, indicating interaction with the phosphatidyl headgroup (kalata B1) and the headgroup and part of the hexanoyl moiety (RTD-1 and oRTD-1), respectively. In the pharmaceutical industry cyclisation is an often used practice to improve the lifetime of peptidic drugs and render them stable against digest by exoproteinase. Circular peptides in general and cyclotides in particular provide an attractive natural scaffold onto which desired functionalities can be grafted or whose natural bioactivities can be altered to our advantage. This study has discovered new sources for circular, disulfide rich peptides and investigated some of their properties, providing a baseline for their possible future use as pharmaceuticals.