The broad aim of this thesis is to generate fundamental knowledge about the structure and function of cyclotides, which are a topologically unique family of proteins. A long-term goal is to use the fundamental knowledge to assist in the development of drugs based on the stable cyclotide framework. Cyclotides are small proteins that are characterised by a cyclic cystine knot (CCK) motif, which is defined as a circular backbone combined with a cystine knot core. So far cyclotides have been found in plants of the Violaceae (violet) and Rubiaceae (coffee) plant families, and are believed to have a defence-related function. From an application perspective, the CCK framework has potential as a drug scaffold, being an ultra-stable alternative to linear peptide models. The reasons why cyclotides show promise as a drug template are three-fold – they have naturally high sequence diversity, suggesting that their framework can accommodate a range of epitopes; they are remarkably stable under various chemical, enzymatic and thermal conditions, which means that they have increased bioavailability; and they have a diverse range of bioactivities, supporting the notion that they can be used in a number of therapeutic applications. These three reasons are intimately linked to three core knowledge domains of cyclotide research, namely cyclotide sequences, structures and interactions. Thus, fundamental research into these three domains, as investigated in this thesis, is important as it may assist in the development of drugs based on the CCK scaffold. Chapter 1 of this thesis provides the background information to define the molecules studied and to highlight their importance. Chapter 2 describes the main experimental techniques that were used in this thesis, including nuclear magnetic resonance spectroscopy and mass spectrometry. The development of the CCK technology may benefit from a thorough understanding of the natural diversity of cyclotide sequences and the significance of this diversity on activity. Chapter 3 reports on the discovery of cyclotides in Viola yedoensis, a Chinese violet that is interesting because it is widely used in Traditional Chinese Medicine to treat a number of illnesses including swelling and hepatitis. In this study, a total of eight cyclotides was characterised, including five novel sequences. Based on anti-HIV and haemolytic assays, a strong relationship between surface hydrophobicity and activity was established. The stability of cyclotides, which underpins their potential as a drug scaffold, is examined at a structural level in Chapter 4. The solution structure of varv F, a cyclotide from the European field pansy, Viola arvensis, was solved and compared to the crystal structure of the same peptide, confirming the core structural features of cyclotides responsible for their stability, including the topology of the cystine knot, which has previously attracted some debate. From a comparison of biophysical measurements of a representative group of five cyclotides, a conserved network of hydrogen bonds, which also stabilises the cyclotide framework, was defined. A subset of hydrogen bonds involving the highly conserved Glu in loop 1 of cyclotides was examined in more detail by solving the structure of kalata B12, the only naturally occurring cyclotide with an Asp instead of a Glu in loop 1. By comparison with the prototypical cyclotide kalata B1 and an Ala mutant E7A-kalata B1, it was shown that the highly conserved Glu is important for both stability and activity. Chapter 5 reports on studies that add to our understanding of the mechanism of action of cyclotides, which is believed to involve membrane interactions. Spin-label experiments were performed for two cyclotides, kalata B2 and cycloviolacin O2, which are representative cyclotides from the two cyclotide sub-families, Möbius and bracelet, respectively. This study showed that different cyclotides have different but very specific binding modes at the membrane surface. Currently, it is believed that for Möbius cyclotides at least (e.g. kalata B1 and kalata B2), self-association may lead to the formation of membrane pores. Oligomerisation of cyclotides was also studied in this chapter using NMR relaxation. A computer program, NMRdyn, was developed to extract microdynamic and self-association parameters from NMR relaxation data. This program was used to analyse 13C relaxation data on kalata B1, providing clues about the tetramer structure of kalata B1. Although the three areas of cyclotide research examined in this thesis – sequence, structure and interactions – are reported in separate sections, the areas are not independent of each other. For example, the mechanism of action of cyclotides, which is reported in Chapter 3, requires an understanding of cyclotide structures, which is reported in Chapter 4. Chapter 6 describes a database, CyBase, which integrates sequence/structure/activity data on cyclotides so that relationships between the three areas can be examined. The database also provides tools to assist in discovery and engineering of cyclic proteins. In summary, several key areas that are fundamental to our understanding of cyclotides have been investigated in this thesis, ranging from cyclotide sequence diversity to their mechanism of action. The work described in this thesis represents a significant advance in our current understanding of cyclotides by providing, for example, explanations to their observed structural stability and how they work through interactions with other biomolecules. The information presented in this thesis is potentially useful in facilitating the long-term goal of developing peptide therapeutics based on the stable cyclotide framework.
| Identifer | oai:union.ndltd.org:ADTP/254024 |
| Creators | Conan Wang |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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