This thesis focuses on proteins with unusual topologies, and in particular those rich in cross-linking disulfide bonds or with a circular backbone. Naturally occurring circular proteins were unknown a decade ago but over recent years several families have been discovered. The cyclotide family is by far the largest group of such proteins and is characterised by a circular backbone and six conserved cysteine residues that form three disulfide bonds in a tight core of the molecule. These have been proposed to form a knotted arrangement in which two disulfide bonds and their interconnecting backbone form an embedded ring through which the third disulfide penetrates.
In this study the high-resolution solution structures of the prototypic members of the cyclotide family, kalata B1 and cycloviolacin 01 have been determined. The results provide, in addition to the backbone geometry, information on sidechain orientations, allowing a detailed analysis of local interactions such as sidechainsidechain and sidechain-mainchain hydrogen bonds. This has led to new insight into the stabilising factors of the fold as well as an understanding of the significance of the conservation of individual amino acids in these molecules. In addition the results have been related to NMR monitored pH and temperature titrations and it has been shown that a conserved Glu residue plays a key role in the structure by accepting up to four hydrogen bonds, thereby stabilising a short stretch of helix in cycloviolacin 01 and a turn in kalata B1. Interestingly kalata Bl and several other memebres of the cyclotide family contains a Trp-Pro motif in which the peptide bond preceding the Pro residue adopts a cis conformation.
As part of a strategy to separately delineate the role of cystine knots and circular backbones additional peptides that had one or the other of these features, compared to both in the cyclotides, were examined.
The structural properties of a novel toxin molecule from the Australian funnel-web spider have been characterised. Like the cyclotides this molecule contains a cystine knot motif but it does not have the additional restraint of a circular a backbone. It is here shown that despite carrying four disulfide bonds this molecule is flexible in solution and in fact adopts two distinct conformations. Three dimensional structures were determined for both these conformations.
Another peptide that has been described as head-to-tail cyclic in the literature is microcin J25. This 21 amino acid peptides is, like the cyclotides, remarkably stable and shows high resistance to both enzymatic digestion and thermal unfolding. While these properties have been suggested to arise from its circular backbone this study has revealed that in fact microcin J25 contains an unusual crosslink between the N-terminus and the sidechain of a Glu and it is this arrangement that is responsible for its unique properties.
The final molecule chosen for this study was pyrrhocoricin, which unlike the other peptides, lacks both a circular backbone and cross-bracing disulfide bonds. As shown here this 20 amino acid peptide is highly flexible in solution lacks a well-defined solution structure. However, there are evidence for turn regions in this molecule and Further a pyrrhocoricin analogue in which the backbone has been cyclised by a linker Was.
Peptides are generally of limited use in the biotechnological and pharmaceutical applications due to their poor stability and limited bioavailability. However peptides like the cyclotides and microcin J25 have properties suggesting that they are capable of overcoming these limitations. One possibility is to use such molecules as templates onto which bioactive epitopes can be drafted. Studies providing a full understanding of the structural features and their contribution to the remarkable stability are a prerequisite to the design of novel bioactive molecules.
In summary, this thesis provides a comprehensive analysis of the topological features of a number of stable bioactive peptides, namely twists (cis peptide bonds preceding Pro residues), knots (a knotted arrangement of the disulfide bonds) and rings (a circular backbone).