Biological processes are frequently driven by protein-protein interactions. The number of known protein interactions is much higher than the number of known protein complex structures. To bridge this gap, data-driven protein-protein docking utilizing experimental or theoretical restraints is applied. In this study the PROTIN_ID method for generating theoretical docking restraints is introduced. PROTIN_ID generates residue clusters on the protein surface based on sequence conservation. Compared to WHISCY and CCRXP, PRO TIN_ ID performs equally well or better. Furthermore, PROTIN_ID has user-friendly features such as the ability to improve the quality of sequence alignments, which improves its performance, and automatically utilizing up-to-date sequence data for experimentally determined proteins or homology models to generate theoretical restraints. A webserver version of PROTIN_ID was implemented for the academic community. Statistical analyses of the conservation of interface residues using the latest version of Benchmark4.0 demonstrated that interface residues are more conserved than non-interface residues. The application of spatial clustering of residues is more efficient to exploit the conservation signal of interface residues, resulting in reliable predictions that are better than predictions generated by 'non-clustering' or at random. Theoretical restraints derived from PROTIN_ID were applied to drive docking and compared to ab initio docking, demonstrating that data-driven docking was more successful. Combining theoretical and experimental restraints to drive docking was compared to experimental-data driven docking. It was shown that combined restraints-driven docking improved because of increased interface residue recall, demonstrating that consensus-data is possibly useful for improvement of docking performance.

Production of protein pharmaceutical formulations has been challenging due to the complexity of protein synthesis, purification and physical and chemical instabilities of the proteins. Accordingly, the aim of this research is to assess the effects of spray drying, freeze drying and crystallisation using different concentrations of four excipients (pluronic®F127, cremophor®EL, β-cyclodextrin and inulin) on protein integrity, thermal stability and biological activity. Lysozyme and Bovine serum albumin were chosen as model proteins. The oral administration of protein pharmaceuticals to the systemic circulation has numerous barriers, including, sharp pH gradients, photolytic enzymes and low epithelial permeability. Trying to overcome these barriers, proteins can be encapsulated in niosomes that are known to protect proteins against the surrounding environment. In this study, Pluronic® F-127 and cremophor® EL were used as co-surfactants for niosome preparations. Niosomes of Span™ 65: cholesterol: co-surfactant were prepared for encapsulation of proteins by film hydration method. Lysozyme and insulin were chosen as model proteins for noisome formulations. Unprocessed, spray-dried, freeze-dried and crystallised proteins were characterised by the following methods: thermal analysis using Differential Scanning Calorimetry (DSC), microscopic examination of the protein particles using Scanning Electron Microscopy and spectroscopic analysis employing Fourier Transform Infra-Red (FT-IR). Moisture contents were determined by Karl Fisher titration. Enzymatic assay were used to measure biological activity. The percentage yield of proteins and protein content were also determnined. Morphology and vesicular sizes of the prepared niosomes were investigated by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (ZetaPlus), respectively. The entrapment efficiency of protein in niosomes was determined by complete vesicle disruption using 50:50 isopropanol:buffer, followed by analysis of the resulting solutions by HPLC. Thermal behaviour of the niosomes was investigated using Differential Scanning Calometry (DSC). Protection of proteins against stimulated gastric fluid (SGF) stimulated intestinal fluid (SIF) were also assessed. Spray-dried and freeze-dried forms of proteins with pluronic® F127 better maintained the native conformation, as indicated by enzymatic assay, of lysozyme. The storage stability of unprocessed, spray-dried and freeze-dried lysozyme was studied over a period of 20 weeks at different temperatures. The results suggested that protein samples prepared with pluronic® F127 were more stable after storage. Crystallising lysozyme with (0.05%) of: pluronic®F-127, cremophor®EL and inulin significantly (P<0.05) maintained higher biological activity compared to a high concentration (0.2%) of the same excipients. However, using (0.2%) of ~cyclodextrin in crystallisation medium led to higher (P<0.05) protein activity compared to (0 .05%) β-cyclodextrin. The storage stability of unprocessed and crystallised protein samples, with and without excipients, showed that crystallisation of lysozyme with 0.05% (w/v) of pluronic• F-127 and 0.2% (w/v) of β-cyclodextrin better maintained the protein activity when compared to unprocessed and crystallised protein with and without excipients. The protein niosomes that prepared with different molar ratios of Span n.1 65, cholesterol and co-surfactants showed different encapsulation efficiency. For Span™ 65, cholesterol and cremophor® EL niosomes, they were not able to encapsulate any protein. Whilst, Span™ 65, cholesterol and pluron ic® F-127 formulations successfully produced insulin and lysozyme niosomes. For insulin containing niosomes, the ratio of 64.7 (Span™ 65): 32.3 (cholesterol): 3.0 (pluronic@ F-127) produced the highest protein encapsulation efficiency and the smallest vesicle size of O.74j..1m . For lysozyme containing niosomes, the maximum protein encapsulation was found in 72.75/24.25/3 .00% molar ratio of Span™ 651 cholesterol/pluronic®F-127 niosomes with vesicle size of 648.0IJm. The release study of proteins from the niosomal preparations in simulated gastric fluid (SGF) and simulated intestinal fiuid (SIF) revealed that insulin and lysozyme efflux from the niosomes is a biphasic process. The release of both proteins (insulin and lysozyme) from the niosomal formulations was found to be dependent on vesicle size, vesicle lamilarity, protein encapsulation, the position of the protein within the niosomal lamilarity and niosomal formulation component ratio . In conclusion , protein formulations using pluronic@F-127 increased protein stabilisation. Pluronic® F-127 is a good candidate to be used for protein formulations employing different processing techniques. The overall results show promise for protein drug delivery systems.

No description available.

No description available.

In the work reported here, the minimal sites of interaction between B-Myb and its partner proteins p300 and cyclin D1 were mapped, and the structural properties and features of the interacting regions characterised in detail. A transactivation-associated central region of B-Myb was conclusively shown to bind to a region of p300 corresponding to the TAZ2, zinc ion binding domain. As is common for many transcriptional activation domains, the B-Myb transactivation region is intrinsically disordered in isolation but exhibits coupled folding upon binding p300. The specific B-Myb-p300 complex formed was found to adopt a 1:1 stoichiometry with a measured dissociation constant of less than ~3.0 X 10-8 M. An adjacent region of B-Myb containing part of exon 9a, was also found to interact with the preceeding ZZ region of p300, but substantially weaker than observed for theB-Myb-TAZ2 interaction. These findings clearly show that the conserved cysteine/histidine rich region 3 (C/H3) of p300 provides an extensive binding site for B-Myb.;In parallel work, a large central part of B-Myb containing both the transactivation region and exon 9a was found to show no binding to full-length cyclin D1. Together with previous work, this suggests that residues in the N-terminal DNA binding domain of B-Myb are also involved in cyclin D1 binding. Overall, this work suggests that the inhibition of cooperativity between B-Myb and p300 by cyclin D1 is not due to competition for a common binding site on B-Myb.

Epsl5 homology (EH) domain-containing proteins have been implicated in diverse intracellular signalling pathways, such as endocytosis, actin cytoskeleton organization, nucleo-cytosolic shuttling and mitogenic signalling. However, the extent of the protein-protein interactions mediated by the EH domain at the level of a whole organism has not yet been addressed. This project aims to gain an overview of the EH network in a model system, C. elegans, by identifying interactors of all EH-domain containing proteins present in C. elegans. Five genes encode EH proteins in the C. elegans genome and the isolated EH domains were used to screen a C. elegans cDNA library using the Yeast Two Hybrid system. The validation of the putative interactions was carried out by in vitro pull-down assays. The biological relevance of the interactions was tested genetically using C elegans as a model system. The genetic interactions were monitored using available mutants for four of the five EH encoding genes, in which the genes of the putative interactors were knocked-down by RNA interference.

Wnt signaling is involved in self-renewal and maintenance ofhematopoietic stem cells (HSCs); however, the particular role of non-canonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here I show Flamingo and Frizzled8, members of non-canonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Flamingo regulates Frizzled8 distribution at the interface between HSCs and N-cadherin + osteoblasts (N-cad+OBs that enrich osteoprogenitors) in the niche. I further show that N-cad+OBs predominantly express non-canonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. This non-canonical Wnt signaling is attenuated prior to activation of HSCs. In the activated HSCs, however, canonical Wnt signaling is enhanced. Mechanistically, non-canonical Wnt signaling mediated by Frizzled8 suppresses the Ca2+-NF AT- IFNy pathway and antagonizes canonical Wnt signaling in HSCs. My findings demonstrate that non-canonical Wnt signaling maintains quiescent long- term HSCs through Flamingo and Frizzled8 interaction in the niche.

The correct formation of a stable tertiary structure is essential for most proteins to fulfil their function. Besides the loss of function, the misfolding of the polypeptide chain can lead to aggregation which has a potentially lethal outcome for living systems. Under certain conditions proteins can form highly organised, elongated aggregates called amyloid fibrils. More than 30 human proteins are involved in such diseases which are characterised by the formation of amyloid deposits in vivo. Although several properties of amyloid aggregates are similar, the monomeric proteins that form amyloid fibrils in disease do not share a common characteristic amino acid sequence or secondary structure motif. Only two of the disease-causing proteins, β2-microglobulin (β 2m) and immunoglobulin (Ig) light chain (LC), belong to the same fold family. Whilst the aggregation mechanism of ~2m has been extensively studied, our knowledge of LC polymerisation is limited. Comparison of the molecular changes that lead to aggregation of these structurally related proteins provides a unique opportunity to learn more about amyloid formation. In this thesis the work presented in Chapter 3 describes the development of a high yield expression, purification and refolding protocol to .establish the production of two K LC variable domains, SMA and LEN. These two variants show different thermodynamic stability and ability to form amyloid aggregates despite their highly similar primary sequences. Out of the 8 amino acid differences in their sequences two (P40L and Y96Q) have been shown previously to play crucial roles in destabilising LEN. Together with the wild-type proteins the two single mutants and the double mutant (P40LIY96Q) of LEN were also generated and characterised. After the optimisation of conditions under which amyloid-like fibrils could be generated from these proteins in vitro, 1H_15N HSQC NMR experiments were conducted to investigate the main structural changes that occur under amyloidogenic conditions. Due to the size and insolubility of amyloid fibrils the study of the structural properties of these aggregates by conventional biochemical and biophysical methods is challenging. Moreover, it is even more difficult to characterise structurally amyloid assembly intermediates using structural methods. As a consequence, it is essential to develop new approaches to achieve a better understanding of the structural molecular mechanism of amyloid formation. To address two of the main questions about this phenomenon - what are the conformational changes of monomers during the early aggregation stage and what is the nature of protein- protein interactions within early oligomers/fibres - the use of non-natural amino acids to provide new probes of structure formation was chosen. The studies on this subject are detailed in Chapter 4 and 5. First, by introducing a cross-linking group into the sequence of the monomers side chains proximal to each other within fibrils and oligomers at different aggregation states can be captured and identified. In principal, therefore, this approach could provide more detailed information about the conformation properties of the polypeptide chains during amyloid assembly, as well as within the fibrils themselves. For this project, the non-natural amino acid p-benzoyl-l-phenylalanine (pBpa) was utilised. Chapter 4 of this thesis details the development of a method for the efficient in vivo insertion of pBpa into the sequence of β2m. After designing and generating a library of 20 mutants, the expressed variants were characterised and their ability to forms fibrils of different morphology was assessed. The results showed that the presence of the non-natural amino acid did not affect the folded conformation of the monomer or the ability to form long-straight or worm-like fibrils. The process of the optimisation of the cross-linking protocol and the first results of the cross-linked fibrils are also presented in Chapter 4. Chapter 5 introduces the development of an approach to study the conformational changes of the monomer in situ prior to aggregation. Using the previously optimised expression system for the incorporation of non-natural amino acids, β 2m variants containing p-cyanophenylalanine (CNP) were produced. This non-natural amino acid can be used as a FRET probe together with Trp. Experiments demonstrating that this amino acid pair is sensitive enough to detect the conformation changes of the monomer induced by different solution conditions are discussed, together with the future potentials of this method for measurement of intra- and inter-molecular distances within the fibrils and oligomers. These approaches open up exciting future prospects for the study of the architecture and formation of amyloid aggregates with potential extension to light chains and other amyloid forming proteins.

No description available.

Protein adsorption under physiological conditions was recognised to be the key step in the modulation of biological responses between materials and an osseous environment. Many studies have shown variation of adsorption behaviour on medical materials but most experiments were performed under non-physiological idealised conditions with idealised samples. The aim of this thesis was to develop a method to analyse protein adsorption on real clinically relevant samples under physiologically relevant conditions. The use of fluorescent probes was identified as a methodology which would facilitate analysis under a range of conditions including fully competitive with real samples that required no specialised surface pre-treatment. Fluoresceinthiureidoaminocaproic acid (FTCA) and a sulforhodamine derivative (SR101), were identified as suitable for coupling to proteins. FTCA labelled bovine serum albumin (BSA) was initially used to validate the technique and found to have several advantages over commercially available total protein assays; including greater sensitivity and facilitate its use under competitive conditions. These experiments also confirmed sensitivity to temperature and test media as well as demonstrating that the technique could be used on idealised dense and real porous granular (as used clinically) samples. Conformational changes associated with protein-surface interactions were observed through variation in protein auto-fluorescence and confirmed with CD analysis, therefore, care in selection of appropriate experimental conditions and fluorophore probes was required. Additionally, labelling facilitated the visualisation in differences in the morphological habit of the surface adsorbed protein species. Investigation of differential response in protein exchange with hydroxyapatite (HA) and 0.8wt% silicon substituted hydroxyapatite (SA) with more biologically relevant proteins; such as bone morphogenetic protein-2 (BMP-2), fibronectin (FN) and osteopontin (OPN) individually and/or competitively in phosphate buffered saline (PBS) or minimum Eagles medium (MEM) supplemented with 10 % foetal bovine serum (FBS) demonstrated SA to have a greater capacity to adsorb selected osteogenic proteins under competitive conditions as compared to HA. Particularly interesting was the BMP-2 findings, which highlighted the role of media in promoting BMP-2 adsorption and the conformation sensitivity of traditional ELISA assays giving rise to unreliable results.