Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithography.

No description available.

Dans l’épiderme, les mélanocytes participent à la protection de la peau contre les rayons ionisants du soleil en synthétisant un pigment, la mélanine, dans des compartiments apparentés aux lysosomes appelés melanosomes. La mélanogenèse est un processus séquentiel initié par la production de fibres amyloïdes dont la composante principale est la protéine PMEL. Ces fibres séquestrent la mélanine et permettent l’élimination d’intermédiaires toxiques produits lors de sa synthèse. La mélanogenèse et le phénotype pigmenté sont affectés lorsque le processus de formation des fibres est altéré. Les fibres résultent du clivage de PMEL dans les endosomes précurseurs des mélanosomes mais les protéases impliquées dans ce processus restent peu ou pas caractérisées. Afin de mieux comprendre les mécanismes de formation des fibres amyloïdes dérivées de PMEL, j’ai étudié le rôle de deux protéases : les Bêta-sécrétases BACE1 et BACE2. En combinant des techniques de biochimie, d’immunocytochimie et d’imagerie photonique et électronique, j’ai montré que la perte de l’expression de Bace2 in vivo (souris KO BACE2) ou sa déplétion (siRNA) dans une lignée de mélanocytes inhibent le clivage amyloïdogénique de PMEL et affectent à la fois la formation de fibres de PMEL dans les mélanosomes et la pigmentation. J’ai pu notamment reproduire in vitro le clivage spécifique de PMEL en utilisant une forme recombinante de BACE2. En parallèle, j’ai également étudié le rôle de BACE1 dans la mélanogenèse. Mes résultats indiquent que BACE1, bien que n’étant pas impliquée dans le clivage de PMEL, régulerait la maturation des mélanosomes précoces in vivo et in cellulo, en modulant les contacts entre mélanosomes et réticulum endoplasmique (RE). Dans les mélanocytes, BACE1 est présente dans le RE et interagit avec des protéines impliquées dans les contacts RE-endosomes. Ces contacts seraient cruciaux pour le transfert de molécules nécessaires à la maturation des mélanosomes. L’ensemble de ces résultats démontre un rôle pour chacune des Bêta-sécrétases dans le processus de mélanogenèse, levant le voile sur des processus clés liés à la biogenèse des mélanosomes. Par ailleurs, les fibres de PMEL constituant le modèle le plus abouti de l’amyloïdogenèse physiologique chez les mammifères, ces études pourraient à plus long terme aider à la compréhension de la formation des fibres amyloïdes pathologiques ; notamment dans la maladie d’Alzheimer où l’amyloïdogenèse d’APP est très similaire à celle de PMEL. / In the epidermis, melanocytes synthetize a pigment called melanin, in lysosome-related-organelles called melanosomes, in order to protect the skin against the ionizing radiations of the sun. Melanogenesis is a sequential process initiated by the formation of amyloid fibrils whose principal component is the protein PMEL. Those fibrils sequester the melanin pigment and allow the removal of toxic intermediates formed during its synthesis. Melanogenesis and the pigmented phenotype are affected when the process of fibrils formation is altered. Fibrils come from the processing of PMEL in endosome precursors of melanosomes but the proteases implicated in this process are not well characterized. In order to better understand the mechanisms implicated in the formation of the PMEL amyloid fibrils, I studied the role of two proteases: the Beta-secretases BACE1 and BACE2. Using a combination of biochemical, immunocytochemical methods and photonic and electronic imaging, I have shown that the loss of Bace2 expression in vivo (BACE2 KO mice) or its depletion (siRNA), in a melanocyte cell line, inhibit the amyloidogenic processing of PMEL and affect both the formation of the PMEL fibrils in melanosomes and pigmentation. I could reproduce in vitro the specific cleavage of PMEL by using a recombinant form of BACE2. In parallel, I have also studied the role of BACE1 in melanogenesis. My results indicate that BACE1, even though it is not implicated in PMEL processing, could regulate the maturation of early melanosomes in vivo and in cellulo, by modulating the contacts between melanosomes and endoplasmic reticulum (ER). In melanocytes, BACE1 is present in the ER and interacts with proteins implicated in ER-endosomes contacts. Those contacts would be crucial for the transfer of molecules that are necessary for melanosome maturation. All together those results demonstrate the role of both Beta-secretases in melanogenesis, and reveal key processes involved in melanosome biogenesis. Moreover, because PMEL fibrils are the most completed model of physiological amyloidogenesis in mammals, theses studies could help in the future the understanding of the formation of pathological amyloid fibrils; in particular in the Alzheimer’s disease where the amyloidogenesis of APP is very similar to the one of PMEL.

The thesis deals with the testing of amyloidogenicity of various carbon nanoparticles and polymers. The first part of the thesis provides the theoretical background of amyloidoses, a group of diseases in which proteins are stored in the insoluble form of amyloid. In addition, the theoretical part also deals with a general overview of nanomaterials and the most important methods. Several types of nanomaterials were tested within the thesis, so the part Results and Discussion was divided into two subchapters: 1) Carbon nanospecies and amyloid fibril formation, and 2) Polysaccharides, glycogen modifications and amyloid fibril formation. The first subchapter concerns the testing of four types of carbon nanoparticles (single-walled carbon nanotubes (SWNT), fullerenes (C60), carbon quantum dots (CDs) and nanodiamonds (NDs)). These materials were tested on a model system hen egg white lysozyme (HEWL). Using fluorescence measurements and transmission electron microscopy (TEM), the nanoparticles were ranked from the most to the least amyloidogenic as follows: NDs> control> C60> CDs> SWNT. The second subchapter deals with the effect of selected polysaccharides (glycogen (GG), mannan (MAN), phytoglycogen (PG)) and modified GG on amyloid fibril formation. These materials were tested on the HEWL model system,...

This work investigates the influence of mutations at selected positions on the structure formation of the Alzheimer’s disease peptide amyloid β. Amyloid β is a member of the class of intrinsically disordered proteins that can aggregate into fibrils, which are characterized by a highly stable secondary structure, called cross-β structure. A central contact during fibrillation is the hydrophobic F19-L34 contact, which is located within the core of the cross-β structure. Modifications of this contact are known to influence the local molecular structure whereas the fibril morphology and the cross-β structure remain stable. In contrast, toxicity of amyloid β was completely lost for all previously investigated mutants of F19 and L34. This work characterizes the properties of this contact and answers the question what the minimally tolerated modifications are. To characterize the structure, structure formation process and biological activity of the Aβ variants a set of experiments was carried out. The local structure and dynamics were investigated using NMR experiments focusing on 13C-chemical shift changes and 1H-13C dipolar couplings, respectively. The fibril morphology and cross- β structure was verified by electron microscopy, circular dichroism spectroscopy and X-ray diffraction. Toxicity and biological activity was investigated using complementary cell culture experiments. The work was divided in three parts. First, L34 was substituted with three highly similar amino acids: the isomer isoleucine, valine that is one methylene group shorter but also a branched chain amino acid and the stereoisomer D-leucine. The L34 position proved to be important for the initiation of the structure formation, oligomer stability, fibril growth and the biological activity of amyloid β. These characteristics and properties were highly sensitive also to minor modifications but the different mutants showed no specific but qualitatively similar effects. The second part complemented previous mutation studies of the F19 position. Four new mutants were designed testing mild modification of the F19-L34 contact: phenylglycine and the homophenylalanine (S)-2-amino-4-phenyl-butyric acid change the length of the side chain, cyclohexyl-alanine eliminates the π-aromaticity of the ring system and increases the 3D steric demand, and (1-naphtyl)-alanine increases the 2D steric demand while maintaining the aromaticity. Mutations at the F19 position caused qualitatively similar effects as L34 modifications but proved to have quantitatively greater impact. Furthermore, they showed some specificity as steric constraints caused larger changes than modifications of the ring system. The third part investigates the influence of β-methylamino-L-alanine (BMAA) substitutions at positions F19, S8, and S26. The serine to BMAA substitutions were included because of their potential medical relevance. A F19BMAA substitution caused similar effects like other modifications at this position. Replacement of serine lead to a structural reorientation of the Aβ N-terminus and turn region. Furthermore, the pathways of the cell response changed from mitochondrial activity and plasma membrane integrity to apoptosis and neuronal stress reaction. Summarizing, it could be shown that, although the formation and structure of amyloid β fibrils is robust against different modifications the fibrillation kinetics, local structure and especially biological activity is highly sensitive and to some extend specific to even minor modifications.

No / ß2-Microglobulin (ß2m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven ß-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E¿)), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native ß2m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59¿71 (peptide E) and 59¿79 (peptide E¿) of intact ß2m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59¿71 may be important in the self-association of partially folded ß2m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.

Is it possible to identify cooked, rather than burnt, bone? Mild heating (≤100 °C,1 h) – typical of cooking – does not lead to detectable changes in any biochemical parameter of bone yet measured. If it is only possible to detect charred bone, how is it possible to detect cooking in the archaeological record? In a previous paper (Koon et al., 2003, J. Arch. Sci.), we used a Transmission Electron Microscopy (TEM) based approach to investigate changes in the organization of the bone protein, collagen, as it is heated, using bone from heating experiments and short term burials. The work revealed that mineralized collagen, despite requiring aggressive treatment to gelatinise the protein (e.g. 90 °C, 240+ h), readily accumulates minor damage. We believe that the presence of mineral matrix stabilises the collagen enabling the damage to accumulate, but preventing it from causing immediate gelatinisation. Once the mineral is removed, the damage can be observed using appropriate visualization methods. In this paper the visualization technique was tested in a blind study of bovine bone from the Anglo-Scandinavian site of Coppergate, York. The purpose of the study was to determine if the method could discriminate between bones thought likely, on the basis of zoo-archaeological and spatial evidence, to have been cooked (high meat yield bones from a domestic context) and those which were butchered but unlikely to have been cooked (low yield bones from a butchery site). The results of the TEM analysis identified two clear groups of bones, one set more damaged than the other. This finding was consistent with archaeozoological interpretation, with the exception of one bone from the domestic context, which was not identified as having been cooked.

This dissertation focuses on the solution-state polymer assembly of conjugated polymers with specific attention to nano- and molecular-scale morphology. Understanding how to control these structures holds potential for applications in polymer-based electronics. Optimization of conjugated polymer morphology was performed with three objectives: 1) segregation of donor and acceptor materials on the nanometer length-scale, 2) achieving molecular-scale ordering in terms of crystallinity within distinct domains, and 3) maximizing the number and quality of well-defined donor/acceptor interfaces. Chapter 1 introduces the development of a mixed solvent method to create crystalline poly(3-hexyl thiophene) (P3HT) fibrils in solution. Chapter 2 describes fibril purification and approaches to robust and functional fibrils, while chapters 3 and 4 demonstrate the formation of hybrid nanocomposite wires of P3HT and cadmium selenide (CdSe) nanoparticles by two methods: 1) co-crystallization of free and P3HT-grafted CdSe for composite nanowires and 2) direct attachment of CdSe nanoparticles at fibril edges to give superhighway structures. These composite structures show great potential in the application of optoelectronic devices, such as the active layer of solar cells. Finally, ultrafast photophysical characterization of these polymers, using time-resolved photoluminescence and transient absorption, was performed to determine the aggregation types present in suspended fibrils and monitor the formation and decay of charged species in fibrils and donor-acceptor systems

Light chain (AL) amyloidosis is a disease that occurs due to the presence of a small plasma-cell clone, which produces amyloidogenic light chains. These chains can misfold and aggregate, leading to the deposition of amyloid fibrils in tissues. If left untreated or if treatment is ineffective, this can result in irreversible organ dysfunction and eventual death. Current therapeutic treatments generally target and remove the clonal plasma cell population responsible for secreting full-length light chains which is not always effective or safe, however, a different approach to halt pathological LC misfolding would be to inhibit the amyloidogenesis cascade at its starting point. Small molecules have been identified that have the ability to bind to highly conserved residues in the interface between heavy and light chains which can be used to potentially impede the process of amyloid fibril deposition before the native FL LC can misfold or undergo proteolysis to form amyloid fibrils. To test whether small-molecule kinetic stabilizers are effective in stabilizing light chains, we measured the ability of the small molecule to bind to LCs, and the ability of light chains to aggregate and unfold in the absence and presence of small-molecule. Our findings suggest that the binding of stabilizers to the interface between variable domains of the LC dimer can increase equilibrium stability and decrease the rate of aggregation, thereby delaying the onset of amyloid formation.

no / Low frequency (0.1¿2 Hz) dynamic mechanical analysis on individual type I collagen fibrils has been carried out using atomic force microscopy (AFM). Both the elastic (static) and viscous (dynamic) responses are correlated to the characteristic axial banding, gap and overlap regions. The elastic modulus (¿5 GPa) on the overlap region, where the density of tropocollagen is highest, is 160% that of the gap region. The amount of dissipation on each region is frequency dependent, with the gap region dissipating most energy at the lowest frequencies (0.1 Hz) and crossing over with the overlap region at ¿0.75 Hz. This may reflect an ability of collagen fibrils to absorb energy over a range of frequencies using more than one mechanism, which is suggested as an evolutionary driver for the mechanical role of type I collagen in connective tissues and organs. / BBSRC