Structure and Activity Studies of Cyclotides STRUCTURE AND ACTIVITY STUDIES OF CYCLOTIDES

This thesis focuses on a large family of cyclic mini-proteins, namely the cyclotides, which have been discovered in a range of plants over the past decade. Around 50 cyclotide sequences have been published to date but the family is estimated to comprise hundreds or thousands of members. Most cyclotides consist of approximately 30 amino acids and have a unique and extremely stable three-dimensional structure, combining a cyclic peptide backbone with a knotted arrangement of three disulfide bonds. Cyclotides have a range of biological activities, including uterotonic, neurotensin-antagonistic, anti-cancer, haemolytic, anti-HIV, and insecticidal activities. The natural function of cyclotides in the plant is thought to be defence against plant pathogens and feeding insects. There is considerable current interest in exploring cyclotides as models for development of peptide-based drugs, both because of their stable scaffold and because of their bioactive properties. An important aspect of recent cyclotide research has been to enhance the understanding of how cyclotides exert their biological activities, or their mechanism of action. The broad aims of this thesis were to investigate the relationships between structure and biological activity of the cyclotides, with emphasis on the prototypic cyclotide kalata B1, and to further explore the capabilities of cyclotides or cyclotide analogues as bioactive agents. As cyclotides are very abundant in plants of the Violaceae family, their innate sequence variation was explored for “natural mutagenesis studies” to gain a better understanding of the sequence variation allowed within the cyclotide scaffold, which may be regarded as a natural combinatorial template. Further, the discovery of more cyclotide precursors may help identifying the mechanism by which cyclotides are processed from linear precursors to mature cyclic proteins. PCR-based approaches were used to determine the cDNA sequences of several new cyclotides and cyclotide precursors from Melicytus, Hybanthus and Viola species within the Violaceae family. To develop a timeand cost effective alternative to chemical synthesis for the generation of cyclotide analogues for structure and activity studies, this study explored intein-based bacterial expression systems. Inteins are recently discovered auto-catalytic protein splicing elements that can facilitate splicing and subsequent cyclisation of a target protein expressed as part of an intein-fusion protein. Several kalata B1-intein fusion constructs were cloned and expressed in E. coli cells. Although the yields of cyclised kalata B1 were too low for producing useable amounts of cyclotide mutants for structure and