Analysis and characterisation of biomolecules using mass spectrometry has advanced over the past decade due to improvements in instrument design and capability; relevant use of complementary techniques; and available experimental and in silico data for comparison with cutting-edge research. This thesis presents ion mobility data, collected on an in-house modified QToF mass spectrometer (the MoQTOF), for a number of protein systems. Two haemoproteins, cytochrome c and haemoglobin, have been characterised and rotationally-averaged collision cross-sections for a number of multimeric species are presented. Intact multiply-charged multimers of the form [xCyt c + nH]z+ where x = 1 (monomer), x = 2 (dimer) and x = 3 (trimer) for cytochrome c have been elucidated and for species with x ≥ 2, reported for the first time. Fragment ions possibly attributed to a novel fragmentation mechanism, native electron capture dissociation, are reported with a brief discussion into their possible production from the dissociation of the gas-phase dimer species. Haemoglobin monomer globin subunits, dimers and intact tetramer have been successfully transferred to the gas phase, and their cross-sections elucidated. Comparisons with in silico computational data have been made and a discussion of the biologically-active tetramer association/dissociation technique is presented. Three further proteins have been studied and their gas-phase collision cross-sections calculated. Two regions of the large Factor H (fH) complement glycoprotein, fH 10-15 and fH 19-20, have been characterised for the first time by ion mobility-mass spectrometry. Much work using nuclear magnetic resonance spectroscopy has previously been achieved to produce structural information of these protein regions, however further biophysical characterisation using mass spectrometry may aid in greater understanding of the interactions these two specific regions have with other biomolecules. The DNA-binding core domain of the tumour suppressor p53 has been characterised and cross-sections produced in the presence and absence of the zinc metal ion that may control the domain’s biological activity. Within this core domain, p53 inactivation mutations have been shown to occur in up to 50% of human cancers, therefore the potential exists to further cancer-fighting activity through research on this region. Anterior Gradient-2 (AGR2) protein facilitates downregulation of p53 in an as yet unclear mechanism. Recent work using peptide aptamers has demonstrated that this downregulation can be disrupted and levels of p53 restored. Collision cross-sections for six peptide aptamers have been calculated, as well as cross-sections for multimers of AGR2 protein. A complex between one aptamer with the protein has also been elucidated. Use of the commercially available Synapt HDMS ion mobility-mass spectrometer at Waters MS Technologies Centre (Manchester, UK) allowed data to be collected for both Factor H protein regions and for the DNA-binding core domain of p53. Data are compared in the appropriate chapters with data collected using the MoQTOF.

The work presented in this thesis has contributed with knowledge to several aspects of protein-protein interaction involving PDZ domains. A substantial amount of our proteome contains regions that are intrinsically disordered but fold upon ligand interaction. The mechanism by which disordered regions bind to their ligands is one important piece of the puzzle to understand why disorder is beneficial. A region in the PDZ domain of nNOS undergoes such a disorder-to-order transition to form a b-sheet in the binding pocket of its partner. By studying the kinetics of interaction, in combination with mutations that modulate the stability of the aforementioned region, we demonstrate that the binding mechanism consists of multiple steps in which the native binding interactions of the b-sheet are formed cooperatively after the rate-limiting transition state. These mechanistic aspects may be general for the binding reactions of intrinsically disordered protein regions, at least upon formation of β-sheets.               The second part of this thesis deals with the engineering of proteins for increasing affinity in protein-protein interaction. Infection by high-risk human papillomavirus (hrHPV) can lead to cancer, and the viral E6 protein is an attractive drug target. E6 from hrHPV natively interacts with the well-characterized PDZ2 domain in SAP97, which we used as a scaffold to develop a high affinity bivalent binder of hrHPV E6. We initially increased PDZ2's affinity for E6 6-fold, but at the cost of decreased specificity. Attaching a helix that binds E6 at a distant site, increasing the affinity another14-fold, completed the design.             The final work of this thesis investigates if binding studies conducted with isolated PDZ domains is representative of the full-length proteins they belong to. It has been suggested that ligand binding in PDZ domains can be influenced by factors such as adjacent domains and interactions outside of the binding pocket. We studied these aspects for the three PDZ domains of PSD-95 and found that they on the whole function in an independent manner with short peptides as ligands, but that interactions outside of the PDZ binding-pocket may be present. The representative length of the PDZ interaction partner should therefore be considered.

Overlapping genes are adjacent genes that share a portion of their coding sequence. Such genes are often observed in the compact genomes of viruses, prokaryotes,and mitochondria. Overlapping genes are also seen in human and other mammalian genomes. Gene overlapping is a phenomenon to minimize genomic size and maximize encoding capacity. Overlapping genes produce different proteins. A major task in the post genomic era is the large-scale study of the structures and functions of proteins. Proteins play crucial roles in virtually all biological processes. In general it is assumed that 3-D structure determines the function of proteins, but many proteins or region of proteins may function in the absence of 3-D structure. The term disordered is used to describe these proteins. A large number of studies has shown that biological functions depend on both ordered and disordered proteins. Natively disordered regions are common and play essential roles in many proteins, especially, with regard to activities involved in signaling and regulation. The goal of this research was the analysis of the ordered and disordered tendencies of viral proteins encoded by overlapping genes. Our hypothesis is that, in a pair of proteins or protein regions encoded by overlapping genes, at least one of the pair is disordered (or unstructured). Our hypothesis is based on the observation that structural proteins require highly specific amino acid sequences, while unstructured (disordered) sequences are essentially unconstrained. Thus, given a structural protein and its associated mRNA sequence, any sequence derived from an overlapping reading frame seems highly unlikely to have a sequence pattern commensurate with a structural protein; on the other hand, a sequence pattern consistent with a disordered protein seems much more likely. We performed studies on the protein products of overlapping gene sequences, tested the hypothesis and addressed the following two questions: First do the proteins encoded by overlapping genes have opposite order-disorder content, that is, does the ordered part of one of the overlapping proteins correspond to a disordered part in the other overlapping protein? Second, does the encoded protein in the overlapping regions have more disordered amino acids than the non-overlapping regions? Using our database of overlapping viral genes and the protein predictor PONDR VL3, we predicted the order-disorder of amino acids in the sequence of 97 viral protein samples. An analysis of the results supported our hypothesis and indicated that the ordered amino acids are mostly associated with non-overlapping regions while disordered amino acids are more prevalent in overlapping regions. In the overlapping regions for 52 protein pairs, we showed that most of the amino acid pairs facing each other on the protein sequences had at least one disorder for most cases. Out of 52 pairs, there were 3 protein pairs where there were no disordered amino acids and 22 protein pairs where there were no ordered amino acids on either sequence. The fraction of ordered pairs in the pool of overlapping regions of 52 protein pairs was 0.28. The non-overlapping region of 97 proteins had predominantly ordered proteins. The fraction of ordered amino acids in the pool of non-overlapping regions was determined to be 0.77.