Characterisation of protein structure and interactions: novel applications to the study of bioactive peptides.

Abstract

The studies of protein/peptide folding, misfolding, structure, and interactions are vital to understanding complex biological problems. The work presented in this thesis describes the development and application of a variety of biophysical techniques to investigate protein structure and interactions, with applications to the structure and function of several bioactive peptides. Firstly, the development of a novel negative ion amenable chemical crosslinking-mass spectrometry (CX-MS) approach is described. CX-MS is a low-resolution technique to study protein structure and interactions. It involves covalent modification and tethering of a protein complex by a reactive reagent, followed by proteolytic digestion. The sites of the intra- and inter-molecular crosslinks provide distance restraints for modelling and enables conclusions to be drawn about the three-dimensional structure and binding interfaces within a protein complex. However, easy identification of crosslinks amongst the large quantity of proteolytic fragments remains challenging. In this study, the application of novel disulfide-based MS cleavable crosslinking reagents was investigated as a tool to easily identify crosslinked peptides by their highly reproducible and characteristic fragmentation patterns in the negative ion mode. MS3 analysis of the product anions allows easy sequencing and identification of crosslinking sites. Preliminary investigations validate these reagent as a tools to readily identify chemical crosslinks within proteins and their complexes, demonstrating that this approach is an effective and efficient means to determine aspects of the topologies of protein complexes of biological importance. Secondly, the use of several biophysical methods is described to probe the structures of a variety of complexes involving the regulatory protein calmodulin (CaM) with bioactive amphibian peptides. CaM is ubiquitous in nature and plays a regulatory role in numerous biological processes, including some in amphibians and their predators; for example, it is involved in the upregulation of nitric oxide synthesis in vivo. Isothermal titration calorimetry was used to investigate the specific heats of the interactions, ion mobility-mass spectrometry was used to investigate the changes in collision cross section that occur as a result of complexation and nuclear magnetic resonance spectroscopy was used to track chemical shift changes upon binding. The results obtained confirm that these complexes adopt canonical collapsed structures and demonstrate the strength of the interaction between the peptides and CaM. Next, work is presented which investigated the abilities of several bioactive amphibian peptides to inhibit fibril formation by disease related proteins. The peptide caerin 1.8 and several synthetic modifications were tested for their ability to inhibit fibril formation by the Alzheimer’s related amyloid-β (1-42) peptide. The results obtained show that caerin 1.8 redirects the aggregation process of amyloid-β (1-42) toward the amorphous aggregation pathway. In addition, the self-assembly properties of the antimicrobial peptide uperin 3.5 were investigated using a variety of biophysical techniques, including transmission electron microscopy, ion mobility-mass spectrometry, circular dichroism, thioflavin T binding and cell viability assays. Similarities were observed between the fibrils formed by this peptide and those of disease related proteins, supporting the notion that information can be obtained about disease related amyloid fibril formation by studying amyloidogenic host-defence peptides. Lastly, work detailing the effect of aspartic acid (Asp) isomerisation to isoAsp on the structure, activity and proteolytic cleavage susceptibility of three amphibian peptides, Crinia angiotensin II, uperin 1.1 and citropin 1.1 is presented. isoAsp formation has been shown to occur naturally as a result of age-related protein degradation, and is a consideration when preparing formulations of peptide therapeutics. isoAsp formation causes a ‘kink’ in the normally helical structure of citropin 1.1, as determined by nuclear magnetic resonance spectroscopy, which results in a reduction of its antimicrobial activity. The effect of this isomerisation process on the smooth muscle activities of Crinia angiotensin II and uperin 1.1 was different, with Asp isomerisation in Crinia angiotensin II causing a decrease in activity, and Asp isomerisation in uperin 1.1 causing greater contraction at lower concentrations. Proteolytic cleavage with trypsin was identical for each pair of Asp/isoAsp isomers, whilst cleavage with α-chymotrypsin was different for the two Asp/isoAsp citropin 1.1 isomers due to the presence of isoAsp adjacent to the cleavage site.