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Protein aggregation has been implicated in several catastrophic diseases (neurodegeneration, diabetes, ALS) and its complexity has also become a major obstacle in large-scale production of protein-based therapeutics. Despite the generic behavior of proteins to aggregate, only a few globular proteins have known aggregation mechanisms. At present, there have been no clear connections between a protein folding, function and aggregation. We have tackled the challenge of understanding the links between a protein's natural tendency to fold and function with its propensity to misfold and aggregate. Using a predominantly beta-sheet protein whose in vitro folding has been explored in detail: cellular retinoic acid-binding protein I (CRABP 1), as a model, we investigated sequence determinants for folding and aggregation. In addition, we characterized the aggregation-prone intermediate under native conditions. Our studies revealed similar contiguous aggregation cores in in vitro and in vivo aggregates of CRABP 1 validating the importance of sequence information under extremely different conditions. Hydrophobic stretches that comprise the interface in aggregates include residues surrounding the ligand binding portal and residues at the C-terminal strands of CRABP 1. Folding studies reveal that docking of the N and C terminals happen in the early stages of barrel closure of CRABP 1 emphasizing the role of folding in preventing exposure of risky aggregation-prone sequences. We further examined the intermediate that initiates aggregation under native conditions. We found that inherent structural fluctuations in the native protein, relevant to ligand binding of CRABP 1, expose aggregation-prone sequences. Binding of the ligand, retinoic acid decreases the aggregation of CRABP 1 illustrating the contribution functional interactions in avoiding aggregation. Our study implies that because of the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable risk.

Nothing is perfect, and this includes the ability to maintain homeostasis within the cell with age. Factors such as aging, chemicals, and gene dysfunction disrupt cellular homeostasis, leading to increased stress and compromising the ability of animals to maintain a healthy lifespan. Dysregulated homeostasis can be detrimental on an organismal level, impacting locomotion, and on a cellular level causing proteins to misfold and become aggregates, which are toxic to cells. Toxic protein aggregation and loss of locomotory function are key hallmarks of several age-related diseases. My Ph.D. work examined the collapse of homeostasis on electrotaxis, the age-associated increase in proteotoxicity, the decline in longevity, and neuronal and muscle health. On a behavioural level I demonstrated that loss of various components of the MT-UPR, ER-UPR, and HSR modulated the speed of animals. Additionally, I found that activation of stress responses due to chemicals and exercise reduced and increased the speed of animals respectively. On a cellular level I elucidated potential mechanisms by which Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF) affects the stress response to maintain homeostasis and prevent protein aggregation. I observed the novel localization and role of MANF in lysosomes to potentially act as a critical regulator of the stress response to maintain proteostasis, neuronal health and longevity, thereby bringing balance to the cell. Furthermore, the broad tissue expression of MANF revealed its localization to muscles. This supports the ability of MANF to act as more than a neurotrophic factor as it was found to be required for muscular health in animals in an age-dependent manner. Overall, my Ph.D. research has provided new insights into the stress response and behaviour and the precise role of MANF in mediating stress response signaling to promote organismal and cellular fitness. / Dissertation / Doctor of Science (PhD) / Cellular perturbations or stress disrupt homeostasis, activating multiple stress responses. Activation of the stress response can determine the fate of an organism and is crucial to its health. Although the stress response pathways are generally understood, little is known about how the stress responses preserve animal behaviour or how they are regulated to promote organismal survival. My work has provided a basis for how stress responses affect behaviour positively and negatively in animals. I found that the stress response required mesencephalic astrocyte derived neurotrophic factor (MANF) to promote organismal survival. My thesis determined that MANF acts as more than a neurotrophic factor. MANF was found to not only be essential in neuronal health but also longevity and muscle health. Overall, this thesis demonstrated the impact of stress response on behaviour and the potential mechanism by which MANF is cytoprotective in whole organisms.

Proteína dissulfeto isomerase (PDI) é uma ditiol-dissulfeto óxido redutase ubíqua que é responsável por uma série de funções celulares, inclusive na sinalização celular e nas respostas a eventos que causam dano celular. Entretanto, a PDI pode se tornar disfuncional através das modificações pós-traducionais, incluindo as promovidas por oxidantes biológicos. Estes oxidantes são provavelmente os responsáveis pelas modificações oxidativas pós-traducionais da PDI que foram detectadas em várias condições associadas ao estresse oxidativo, levando à disfunção da proteína. Devido a falta de estudos cinéticos com a PDI nativa e a falta de caracterização dos produtos dessas reações, investigamos se a diminuição da fluorescência da PDI nativa pode ser empregada para estudos da cinética de oxidação com peróxido de hidrogênio. Posteriormente, investigamos a cinética e os produtos da reação entre PDI e peroxinitrito. Nossos experimentos mostraram que a oxidação por excesso de peróxido de hidrogênio levava a uma diminuição da fluorescência de forma dependente do tempo e da concentração do oxidante, permitindo a determinação da constante de velocidade de segunda ordem (k = (17,3±1,3) M-1 s-1, pH 7,4, 25 ºC). Relevantemente, mostramos que o processo era totalmente revertido por DDT, mostrando que o peróxido de hidrogênio oxida quase que exclusivamente os grupos ditióis da PDI (Cys53 e Cys56 e Cys397 e Cys400). Utilizando a mesma abordagem para estudar a oxidação da PDI por peroxinitrito, notamos que o decréscimo da fluorescência intrínseca da PDI nativa e a velocidade só era proporcional à concentrações sub-estequiométricas ou estequiométricas do oxidante em relação aos tióis reativos da PDI. Somente nessas condições o processo se mostrava reversível por DDT, indicando que os ditióis da PDI eram o alvo preferencial do peroxinitrito mas que a oxidação de outros resíduos também ocorria. A reação dos tióis reativos da PDI com peroxinitrito foi considerada relativamente rápida (6,9 ± 0,6 × 104 M-1 s-1, pH 7,4, 25 °C), e os resíduos de Cys reativos dos domínios a e a\' aparentam reagir com constantes de velocidade similares. Experimentos de proteólise limitada, simulações cinética e análises de MS e MS/MS confirmaram que o peroxinitrito oxida preferencialmente os tióis redox ativos da PDI para os ácidos sulfênicos correspondentes, que, subsequentemente, reagem com os tióis vizinhos, produzindo dissulfetos (Cys53- Cys56 e Cys397- Cys400). Entretanto, uma fração de peroxinitrito decai para radicais levando à hidroxilação e nitração de outros resíduos próximos ao sítio redox ativo (Trp52 Trp396 e Tyr393). Assim, investigamos também a oxidação da PDI por excesso de peroxinitrito em relação aos grupos tióis reativos por diferentes metodologias. Experimentos de SDS-PAGE, western-blot e atividade redutase mostraram que o peroxinitrito promove inativação, nitração e agregação da PDI de forma dependente da concentração de peroxinitrito. Análises de MS e MS/MS mostraram que, em excesso, o peroxinitrito promove nitração (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) e hidroxilação (Trp52, Trp396) da PDI. Em síntese, nossos estudos contribuem para melhor compreensão da oxidação da PDI por peroxinitrito e de suas possíveis consequências biológicas. / Protein disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, including in cellular signaling and responses to cell-damaging events. Nevertheless, PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants. These oxidants are likely responsible for the oxidative post-translational modifications of PDI, which have detected under various conditions associated with oxidative stress, leading to protein dysfunction. However, the kinetics of the reactions of PDI with biological oxidants received limited studies and the products of these reactions were not characterized. Here, we examined whether the decrease in PDI fluorescence can be employed to follow the kinetics of the reaction of the full-length protein with biological oxidants. Also, we investigated the kinetics and products of the reaction between PDI and peroxynitrite. Our experiments showed that oxidation by excess hydrogen peroxide led to a decrease of PDI intrinsic fluorescence in a time- and concentration-dependent manner , permitting the determination of the second-order rate constant of the reaction (k = (17.3 ± 1.3 ) M1 s-1, pH 7.4, 25 ° C). The oxidation was reversed by DDT, indicating that hydrogen peroxide oxidizes mainly PDI dithiols (Cys53 and Cys56 and Cys397 and Cys400). Using the same approach to study PDI oxidation by peroxynitrite we noted that the decrease of the native PDI fluorescence was proportional to sub-stoichiometric or stoichiometric concentrations of the oxidant relative to that of PDI reactive thiols. Only under these conditions, PDI oxidation was reversed by DDT, indicating that PDI dithiols were the preferred target of peroxynitrite but that oxidation of other residues also occurred. The reaction of the active redox thiols of the PDI with peroxynitrite can be considered relatively fast (6.9 ± 0.6 × 104 M-1 s-1, pH 7.4, 25 ° C), and the reactive Cys residues of domains a and a\' were kinetically indistinguishable. Limited proteolysis experiments, kinetic simulations, and MS and MS/MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which subsequently react with the resolving thiols to produce disulfides (Cys53-Cys56 and Cys397-Cys400). However, a fraction of peroxynitrite decays to radicals leading to hydroxylation and nitration to other residues located close to the active site (Trp52 Trp396 and Tyr393). SDS-PAGE, western blotting and inhibition of the reductase activity experiments confirmed that excess peroxynitrite promotes further PDI oxidation, nitration, inactivation and aggregation in a concentration-dependent manner. MS and MS/MS analyzes showed that peroxynitrite in a ten times excess relative to PDI reactive thiols promote PDI nitration (Tyr43, Tyr49, Tyr196, Tyr393, Trp52, Trp396) and hydroxylation (Trp52, Trp396). In conclusion, our studies contribute to a better understanding of PDI oxidation by peroxynitrite and its possible biological consequences

The misfolding and aggregation of proteins is closely associated with more than fifty human disorders, including Alzheimer's and Parkinson's diseases, all of which are currently incurable and many represent a major threat to human life. The mechanism of protein aggregation is subject to extensive studies. The damaging effects associated with protein aggregation have been attributed to amyloidogenic species that are present during the misfolding process. In particular, oligomeric species are, however, intrinsically difficult to study as a consequence of their low abundance and highly heterogeneous nature. The first chapter of my thesis gives an introduction into the field of protein folding and misfolding with a focus on the study of protein aggregation, and toxic effects relevant to human disorders. The second chapter of my thesis describes the development of a methodology that enables the study of aggregate induced lipid bilayer permeability, possibly the most general mechanism of protein aggregate toxicity. Surface-tethered lipid vesicles functioning as optochemical probes sensitive to membrane integrity are imaged using total internal reflection microscopy. It is shown that oligomeric species of the 42-residue form of the Aβ peptide (Aβ42) are responsible for the membrane disruption. The methodology can be applied to the study of other proteins such as α-synuclein and tau, and the ability of antibodies and chaperones to counteract the aggregate induced lipid bilayer permeability can be assessed. Furthermore, lipid bilayer permeability induced by aggregates formed in human induced pluripotent stem cells can be studied. The third chapter presents a new approach for the measurement of protein aggregation kinetics by following the development of the lipid bilayer permeability over the course of the aggregation process of Aβ42. The aggregation kinetics can be modulated with molecular chaperones and pre-formed seed fibrils, which allows secondary nucleation to be identified as the process that drives the formation of species responsible for the lipid bilayer permeability. The fourth chapter describes the development of a three-pronged strategy to study the mechanism of α-synuclein amyloid formation. The aggregation is studied in the presence of lipid vesicles or pre-formed fibrils at neutral or acidic pH of the solution. The influence of single-point mutations on the aggregation of α-synuclein is described. Furthermore, the strategy is applied to the characterisation of the ability of antibodies and small molecules to inhibit the aggregation, and thus has the potential for the development of therapeutical agents. The work presented in the fifth chapter characterises the amyloid fibril populations formed by α-synuclein and mutational variants associated with familial Parkinson's disease. X-ray crystallography, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy and atomic force microscopy have all been applied to the analysis of these amyloid fibrils. Finally, the sixth chapter summarises the results described in this thesis and points out future opportunities in the context of fundamental and translational studies related to the research area of protein misfolding disorders.

De nombreuses études ont mis en évidence l’importance du contrôle qualité des protéines, c’est à dire des mécanismes de reconformation (chaperons moléculaires) et de dégradation (autophagie, proteasome) des protéines dans différentes pathologies musculaires telles que la dystrophie musculaire d’Ullrich (UCMD), de Duchenne (DMD) ou d’Emery-Dreifuss (EDMD) ; cependant, à l’heure actuelle, aucune n’a été menée sur l’ensemble de ces mécanismes dans un seul et même modèle et sur des cellules musculaires avant leur différenciation en muscles. Nous nous sommes donc intéressés à la fonctionnalité des mécanismes de Contrôle Qualité des Protéines et à leurs interconnexions dans des myoblastes immortalisés de donneurs sains ou de patients atteints de DMD. Nous avons observé une augmentation de l’agrégation protéique dans les cellules DMD. Ce phénomène s’accompagne d’une dérégulation des mécanismes de séquestration par les chaperons moléculaires, conséquence d’une modulation de l’expression des protéines HSPB5 et HSPB8. Les mécanismes de dégradation sont également dérégulés; en effet, nous avons observé d’une part, une diminution de l’activité enzymatique du protéasome ainsi que des molécules d’adressage des protéines multiubiquitinées au protéasome et d’autre part, une augmentation de l’activité du facteur de transcription NF?B, de l’expression de protéines intervenant dans l’autophagie et des complexes BAG3/HspB8 conduisant à une augmentation du flux autophagique. L’ensemble de ces dérégulations reflète l’existence d’un stress d’agrégation protéique dans les myoblastes issus de patients DMD. Dans ce contexte, la modulation pharmacologique du PQC dans ces cellules pourrait représenter une nouvelle stratégie thérapeutique pour la Dystrophie Musculaire de Duchenne / Various studies have highlighted the importance of Protein Quality Control (PQC), including protein refolding (molecular chaperones) and degradation (autophagy, proteasome) mechanisms in inherited muscle disorders such as Ullrich Congenital Muscular Dystrophy (UCMD), Duchenne Muscular Dystrophy (DMD) or Emery-Dreifuss Muscular Dystrophy (EDMD); however, to date, no extensive study has been conducted on these mechanisms in a same model, in muscle cells before muscle differentiation. Thus, we were interested in PQC mechanisms functionality and their interconnection in human immortalized myoblasts from healthy donors or patients suffering from DMD. We observed an increase of protein aggregation in DMD cells. This phenomenon is accompanied by a deregulation of sequestration mechanisms by molecular chaperones, reflected by the modulation of HSPB5 and HSPB8 expression. Degradation mechanisms are also deregulated; indeed, we observed on one hand a decrease of proteasome enzymatic activity and multiubiquitinated proteins UPS-adressing molecules and on the other hand, an increase of NF?B transcription factor’s activity, involved in autophagy, and of BAG3/HSPB8 complexes, leading to an increase of the autophagic flux. These PQC defects reflect the existence of a protein aggregation stress in myoblasts coming from DMD patients. In this context, pharmacological modulation of PQC in these cells could represent a new therapeutic strategy for Duchenne Muscular Dystrophy

Proteins play a major role in living systems and present a wide spectrum of functionalities. Many different types of proteins are involved into biological processes, such as the catalysis of biochemical reactions, cellular membrane transport, immune system response and DNA replication. However, some proteins and peptides might become harmful to living organisms; for example, their abnormal aggregation causes neurodegenerative disorders including Alzheimer disease (AD). One of the causes of AD is the presence of amyloid beta peptides Aβ(1-42), Aβ(1-40), which self-assemble into insoluble fibrils and plaques, which surround neuronal cells impeding synapsis. The number of AD patients is increasing, but a cure has not been founded yet. Therefore, it is crucial to investigate the mechanisms underlying amyloid aggregation and screening for compounds able to prevent this irreversible process. Microfluidics permits characterising the physicochemical properties of proteins, investigate their aggregation and study their interactions with other molecules. Chemical kinetics allows studying the microscopic events occurring during protein self-assembly. The combination of these two techniques provides a powerful tool for the identification of compounds inhibiting the aggregation process. In this thesis by using microfluidics, chemical kinetics and other biophysical assays, I have investigated the proteins isoelectric point (pI) and the inhibition of aberrant Aβ(1-42) self-assembly process. Firstly, I describe the development of a microfluidic platform allowing for the measurement of the protein pI, in a gradient-free manner. This approach overcomes a fundamental limitation of convectional techniques that is the achievement of a stable and well-controlled pH gradient. Secondly, I investigate the inhibiting effect of llama nanobodies on Aβ(1-42) aggregation. The findings from this study show that nanobodies target monomeric species with high affinity whereas interactions with fibril surfaces are weak. Finally, I discuss the use of other compounds inhibiting specific nucleation stages. These include the chaperones clusterin and brichos, as well as soot and pure carbon nanoparticles. Importantly, the addition of both chaperones to Aβ(1-42) solutions has an additive inhibitory effect on aggregation. My findings will improve the characterization of the physicochemical properties of proteins as well as providing promising candidates for the inhibition of specific stages of amyloid beta aggregation opening the way to possible cures for AD disease.

The pre-synaptic protein α-Synuclein (αS) is often linked to the pathology of Parkinson’s disease (PD), an age-related neurodegenerative disorder. Lewy bodies, the cytopathological hallmarks of PD, are found to be rich in aggregates of misfolded αS protein. Metal dyshomeostasis has also been linked to PD due to the accumulation of iron in the substantia nigra pars compacta, and diminished copper levels reported in this same region. Metal dyshomeostasis in the brain coupled with oxidative stress can enhance the aggregation of αS. Recently, it was confirmed that mammalian αS is universally acetylated at the N-terminus, a common post-translational modification in humans. The consequences of this modification have been understudied, and it is believed to impart a functional role under physiological conditions with respect to membrane-interactions and protein folding. In an attempt to elucidate the pathological mechanism behind PD with respect to the structural dynamics of the protein, our investigations were focused on physiologically prevalent, N-terminally acetylated αS (NAcαS) and its interaction with the most prevalent redox-active metal ions in the brain (iron and copper) under both aerobic and/or anaerobic conditions. The structural features associated with metal-bound NAcαS differed depending on the iron oxidation states, where under aerobic conditions Feᴵᴵ stabilized an oligomer-locked, anti-parallel right-twisted β-sheet conformation that could potentially impart toxicity to neurons. In contrast, Feᴵᴵᴵ promoted a fibrillar structure rich in parallel β-sheets. N-terminal capping also altered the Cuᴵᴵ coordination sphere and had a dramatic effect on protein aggregation. Parallel studies on NAcαS variants with different site mutations near the putative copper binding sites (ex: H50Q and F4W) indicated that preferential binding shifts upon changes in the side chain residues. In depth analysis of the electron structure of Cuᴵᴵ-bound NAcαS using electron paramagnetic resonance spectroscopy (EPR) revealed a coordination sphere of N3O1 that includes the H50 residue in the wild-type protein that shifts to an O4 coordination sphere at the C-terminus upon Cuᴵᴵ binding to the disease-relevant H50Q variant. Immunoblotting analyses revealed that copper-induced redox chemistry promoted O2-activation and the subsequent formation of dityrosine crosslinks, a post-translational modification identified as a biomarker of PD. EPR-detection of tyrosyl radical formation in the presence of Cuᴵ-bound NAcαS further supported this radical coupling mechanism. Intermolecular crosslinks within the fibrillar core of NAcαS as well as intramolecular crosslinks within the C-terminal region underpin the role of metal-dioxygen chemistry in PD-related pathology. The unique structural features resulting from iron vs copper coordination to NAcαS inspired studies directed at the synergistic effect of each individual metal species as revealed by photo-initiated crosslinking of NAcαS. C-terminal intramolecular tyrosine interactions were mainly impacted by the presence of both metals, which each have binding sites around the same region. These findings emphasize that protein dynamics, metal binding site conformational changes, as well as aggregation pathways can deviate drastically upon N-terminal acetylation of αS and that protein-metal interactions may play a vital role in PD etiology.

Aggregation of the Amyloid β (Aβ) peptide to amyloid fibrils is associated with the outbreak of Alzheimer’s disease. Early aggregation intermediates in form of soluble oligomers are of special interest as they are believed to be the major toxic components in the process. These oligomers are of disordered and transient nature. Therefore, their detailed molecular structure is difficult to access experimentally and often remains unknown. In the present work extensive, fully atomistic replica exchange molecular dynamics simulations were performed to study the preaggregated, monomer states and early aggregation intermediates (dimers, trimers) of Aβ(25-35) and Aβ(10-35)-NH2 in aqueous solution. The folding and aggregation of Aβ(25-35) were studied at neutral pH and 293 K. Aβ(25-35) monomers mainly adopt β-hairpin conformations characterized by a β-turn formed by residues G29 and A30, and a β-sheet between residues N27–K28 and I31–I32 in equilibrium with coiled conformations. The β-hairpin conformations served as initial configurations to model spontaneous aggregation of Aβ(25-35). As expected, within the Aβ(25-35) dimer and trimer ensembles many different poorly populated conformations appear. Nevertheless, we were able to distinguish between disordered and fibril-like oligomers. Whereas disordered oligomers are rather compact with few intermolecular hydrogen bonds (HBs), fibril-like oligomers are characterized by the formation of large intermolecular β-sheets. In most of the fibril-like dimers and trimers individual peptides are fully extended forming in- or out-of-register antiparallel β-sheets. A small amount of fibril-like trimers contained V-shaped peptides forming parallel β-sheets. The dimensions of extended and V-shaped oligomers correspond well to the diameters of two distinct morphologies found for Aβ(25-35) fibrils. The transition from disordered to fibril-like Aβ(25-35) dimers is unfavorable but driven by energy. The lower energy of fibril-like dimers arises from favorable intermolecular HBs and other electrostatic interactions which compete with a loss in entropy. Approximately 25 % of the entropic cost correspond to configurational entropy. The rest relates to solvent entropy, presumably caused by hydrophobic and electrostatic effects. In contrast to the transition towards fibril-like dimers the first step of aggregation is driven by entropy. Here, we compared structural and thermodynamic properties of the individual monomer, dimer and trimer ensembles to gain qualitative information about the aggregation process. The β-hairpin conformation observed for monomers is successively dissolved in dimer and trimer ensembles while instead intermolecular β-sheets are formed. As expected upon aggregation the configurational entropy decreases. Additionally, the solvent accessible surface area (SASA), especially the hydrophobic SASA, decreases yielding a favorable solvation free energy which overcompensates the loss in configurational entropy. In summary, the hydrophobic effect, possibly combined with electrostatic effects, yields an increase in solvent entropy which is believed to be one major driving force towards aggregation. Spontaneous folding of the Aβ(10-35)-NH2 monomer was modeled using two force fields, GROMOS96 43a1 and OPLS/AA, and compared to primary NMR data collected at pH 5.6 and 283 K taken from the literature. Unexpectedly, the two force fields yielded significantly different main conformations. Comparison between experimental and calculated nuclear Overhauser effect (NOE) distances is not sufficient to distinguish between the different force fields. Additionally, the comparison with scalar coupling constants suggest that the chosen protonation in both simulations corresponds to a pH lower than in the experiment. Based on this analysis we were unable to determine which force field yields a better description of this system. Dimerization of Aβ(10-35)-NH2 was studied at neutral pH and 300 K. Dimer conformations arrange in many distinct, poorly populated and rather complex alignments or interlocking patterns which are rather stabilized by side chain interactions than by specific intermolecular hydrogen bonds. Similar to Aβ(25-35) dimers, transition towards β-sheet-rich, fibril-like Aβ(10-35) dimers is driven by energy competing with a loss in entropy. Here, transition is mediated by favorable peptide-solvent and solvent-solvent interactions mainly arising from electrostatic interactions. / Die Aggregation des Amyloid β (Aβ) Peptids zu Amyloidfibrillen wird mit dem Ausbruch der Alzheimer Krankheit in Verbindung gebracht. Die toxische Wirkung auf Zellen wird vor allem den zeitigen Intermediaten in Form von löslichen Oligomeren zugeschrieben. Aufgrund deren ungeordneter und flüchtiger Natur kann die molekulare Struktur solcher zeitigen Oligomere oft experimentell nicht aufgelöst werden. In der vorliegenden Arbeit wurden aufwendige atomistische Replica-Exchange-Molekulardynamik-Simulationen durchgeführt, um die molekulare Struktur von Monomeren und Oligomeren der Fragmente Aβ(25-35) und Aβ(10-35)-NH2 in Wasser zu untersuchen. Die Faltung und Aggregation von Aβ(25-35) wurde bei neutralem pH und 293 K untersucht. Monomere dieses Fragments bilden hauptsächlich β-Haarnadelkonformationen im Gleichgewicht mit Knäulstrukturen. Innerhalb der β-Haarnadelkonformationen bilden die Residuen G29 und A30 einen β-turn, während N27–K28 and I31–I32 ein β-Faltblatt bilden. Diese β-Haarnadelkonformationen bildeten den Ausgangspunkt zur Modellierung spontaner Aggregation. Wie zu erwarten, bilden sich eine Vielzahl verschiedener, gering besetzter Dimer- und Trimerkonformationen. Mit Hilfe einer gröberen Einteilung können diese in ungeordnete und fibrillähnliche Oligomere unterteilt werden. Ungeordnete Oligomere bilden kompakte Strukturen, die nur durch wenige intermolekulare Wasserstoffbrückenbindungen (HBB) stabilisiert sind. Typisch für fibrillähnliche Oligomere ist hingegen die Ausbildung großer intermolekularer β-Faltblätter. In vielen dieser Oligomere finden wir antiparallele, in- oder out-of-register β-Faltblätter gebildet durch vollständig ausgestreckte Peptide. Ein kleiner Teil der fibrillähnlichen Trimere bildet parallele, V-förmige β-Faltblätter. Die Ausdehnungen ausgestreckter und V-förmiger Oligomere entspricht in etwa den Durchmessern von zwei verschiedenen, experimentell gefundenen Fibrillmorphologien für Aβ(25-35). Die Umwandlung von ungeordneten zu fibrillähnlichen Aβ(25-35) Dimeren ist energetisch begünstigt, läuft aber nicht freiwillig ab. Fibrillähnliche Dimere haben eine geringere Energie aufgrund günstiger Peptidwechselwirkungen (HBB, Salzbrücken), welche durch den Verlust an Entropie kompensiert wird. Etwa 25 % entsprechen dem Verlust an Konfigurationsentropie. Der restliche Anteil wird einem Verlust an Lösungsmittelentropie aufgrund von hydrophoben und elektrostatischen Effekten zugesprochen. Im Gegensatz zur Umwandlung in fibrillähnliche Dimere, ist die Assoziation von Monomeren oder Oligomeren entropisch begünstigt. Beim Vergleich thermodynamischer Eigenschaften der Monomer-, Dimer- und Trimersysteme zeigt sich im Verlauf der Aggregation, wie erwartet, eine Abnahme der Konfigurationsentropie. Zusätzlich nimmt die dem Lösungsmittel zugängliche Oberfläche (SASA), insbesondere die hydrophobe SASA, ab. In Verbindung damit beobachten wir eine Abnahme der freien Solvatisierungsenergie, welche den Verlust an Konfigurationsentropie kompensiert. Mit anderen Worten, der hydrophobe Effekt in Kombination mit elektrostatischen Wechselwirkungen führt zu einem Ansteigen der Lösungsmittelentropie und begünstigt damit die Aggegation. Die spontane Faltung des Aβ(10-35)-NH2 Monomers wurde für zwei verschiedene Proteinkraftfelder, GROMOS96 43a1 und OPLS/AA, untersucht und mit primären NMR-Daten aus der Literatur, gemessen bei pH 5.6 und 283 K, verglichen. Beide Kraftfelder generieren unterschiedliche Hauptkonformationen. Der Vergleich zwischen experimentellen und berechneten Kern-Overhauser-Effekt (NOE) Abständen ist nicht ausreichend, um zwischen beiden Kraftfeldern zu unterscheiden. Der Vergleich mit Kopplungskonstanten aus Experiment und Simulation zeigt, dass beide Simulationen einem pH-Wert geringer als 5.6 ensprechen. Basierend auf den bisherigen Ergebnissen können wir nicht entscheiden, welches Kraftfeld eine bessere Beschreibung für dieses System liefert. Die Dimerisierung von Aβ(10-35)-NH2 wurde bei neutralem pH und 300 K untersucht. Wir finden eine Vielzahl verschiedener, gering besetzter Dimerstrukturen, welche eher durch Seitenkettenkontakte als durch spezifische HBB stabilisiert sind. Wie bei den Aβ(25-35) Dimeren, ist die Umwandlung zu β-Faltblattreichen, fibrillähnlichen Aβ(10-35) Dimeren energetisch begünstigt, konkurriert aber mit einem Entropieverlust. Die Umwandlung wird in diesem Fall durch elektrostatische Wechselwirkungen zwischen Peptid und Lösungsmittel und innerhalb des Lösungsmittels bestimmt.

There are many applications of small organic compounds, e.g. as drugs or as tools to study biological systems. Once a compound with interesting biological activity has been found, medicinal chemists typically synthesize small libraries of compounds with systematic differences to the initial “hit” compound. By screening the new ensemble of compounds for their ability to perturb the biological system, insights about the system can be gained. In the work presented here, various ways to synthesize small libraries of ring-fused 2‑pyridones have been developed. Members of this class of peptidomimetic compounds have previously been found to have a variety of biological activities, e.g. as antibacterial agents targeting virulence, and as inhibitors of the aggregation of Alzheimer b‑peptides. The focus in this work has been to alter the core skeleton, the central fragment, of the previously discovered biologically active 2‑pyridones and evaluate the biological effects of these changes. Several new classes of compounds have been constructed and their preparations have included the development of multi-component reactions and a method inspired by diversity-oriented synthesis. Some of the new compounds have been evaluated for their effect on the fibrillation of different amyloid proteins. Both the Parkinson-associated amyloid protein a-synuclein and the bacterial protein CsgA that is involved in bacterial biofilm formation are affected by subtle changes of the compounds’ central fragments. This is an example of the usefulness of central-fragment alterations as a strategy to probe structure-activity relationships, and the derived compounds may be used as tools in further study of the aggregation of amyloid proteins.