Pattern discovery in protein structures is a fundamental task in computational biology, with important applications in protein structure prediction, profiling and alignment. We propose a novel approach for pattern discovery in protein structures using Particle Swarm-based flying windows over potentially promising regions of the search space. Using a heuristic search, based on Particle Swarm Optimization (PSO) is, however, easily trapped in local optima due to the sparse nature of the problem search space. Thus, we introduce a novel fitness-based stagnation detection technique that effectively and efficiently restarts the search process to escape potential local optima. The proposed fitness-based method significantly outperforms the commonly-used distance-based method when tested on eight classical and advanced (shifted/rotated) benchmark functions, as well as on two other applications for proteomic pattern matching and discovery. The main idea is to make use of the already-calculated fitness values of swarm particles, instead of their pairwise distance values, to predict an imminent stagnation situation. That is, the proposed fitness-based method does not require any computational overhead of repeatedly calculating pairwise distances between all particles at each iteration. Moreover, the fitness-based method is less dependent on the problem search space, compared with the distance-based method. The proposed pattern discovery algorithms are first applied to protein contact maps, which are the 2D compact representation of protein structures. Then, they are extended to work on actual protein 3D structures and interaction networks, offering a novel and low-cost approach to protein structure classification and interaction prediction. Concerning protein structure classification, the proposed PSO-based approach correctly distinguishes between the positive and negative examples in two protein datasets over 50 trials. As for protein interaction prediction, the proposed approach works effectively on complex, mostly sparse protein interaction networks, and predicts high-confidence protein-protein interactions — validated by more than one computational and experimental source — through knowledge transfer between topologically-similar interaction patterns of close proximity. Such encouraging results demonstrate that pattern discovery in protein structures and interaction networks are promising new applications of the fast-growing and far-reaching PSO algorithms, which is the main argument of this thesis. / Thesis (Ph.D, Computing) -- Queen's University, 2014-04-21 12:54:03.37

The research described in this thesis employs a variety of Electron Paramagnetic Resonance (EPR) techniques for the study of the electronic and structural properties of artificial supramolecular porphyrin systems and of protein complexes of biological relevance. The electron delocalisation in the cationic radical and photoexcited triplet states of linear and cyclic Π-conjugated multiporphyrin arrays was investigated. In the radical cations, information on the extent of delocalisation can be inferred from the measurement of hyperfine couplings, either indirectly from the continuous wave EPR spectrum or directly using pulsed hyperfine EPR techniques. The results of room temperature EPR experiments showed complete delocalisation of the electron on the timescale of the EPR experiments, but frozen solution EPR measurements revealed localisation onto mainly two to three porphyrin units in the larger porphyrin systems. Information on the delocalisation of the triplet state in the same porphyrin systems is contained both in the hyperfine couplings and in the zero-field splitting (ZFS) interaction. The results outlined in this thesis show that the hyperfine couplings provide a much more accurate estimate of the extent of delocalisation. The trends in proton and nitrogen hyperfine couplings with the size of the porphyrin systems indicate uneven spin density distributions over the linear arrays, but complete delocalisation in the cyclic systems. Time-resolved EPR and magnetophotoselection experiments have shown a reorientation of the zero-field splitting tensor between a single porphyrin unit and longer linear arrays, resulting in alignment of the main optical transition moment and the Z axis of the ZFS tensor. Continuous wave and pulsed dipolar EPR techniques were employed for the determination of the structure of two different protein complexes, the homomultimeric twin-arginine translocase A (TatA) protein channel and the ferredoxin-P450 complex of the electron transport chain in Novosphingobium aromaticivorans. The interaction between nitroxide spin labels introduced at different positions of the TatA monomer was investigated in the complex reconstituted in detergent micelles by analysing the dipolar broadening of the EPR spectra and the results of three- and four-pulse Double Electron-Electron Resonance (DEER) measurements. In combination with results from NMR and molecular dynamics calculations, a structure for the channel complex was proposed. The structure of the ferredoxin-cytochrome P450 complex was investigated by orientation-selective DEER between nitroxide labels introduced on the cytochrome P450 protein and the iron-sulfur cluster of the ferredoxin. The distance and orientation information contained in the experimental DEER data was interpreted in terms of a structural model of the protein complex by orientation-selective DEER simulations combined with a modelling approach based on protein-protein docking.

Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...

A catepsina L é uma cisteína proteinase da família da papaína (clã CA, família C1), sendo esta família a mais conhecida entre as cisteína proteinase. A catepsina L, como outras proteinases da família C1, é sintetizada como uma pró-enzima inativa que é ativada através da remoção do pró-peptídeo. Os pró-peptídeos das catepsinas da subfamília catepsina L apresentam um motivo consenso, denominado motivo ERFNIN. A catepsina L corresponde a principal proteinase digestiva em Tenebrio molitor. No nosso laboratório 3 pró-catepsinas L (pCALs) foram clonadas e seqüenciadas a partir de uma biblioteca de cDNA de intestino médio de larvas de T. molitor: pCAL1 (CAL lisossomal), pCAL2 e pCAL3 (enzimas digestivas). Estas proteinases apresentam o motivo ERFNIN e os resíduos envolvidos na catálise: Cys25, His169, e Asn175 com Gln19 (numeração da papaína). Neste trabalho descrevemos a clonagem em vetores de expressão e a expressão em bactérias das sequências codificadoras de pCAL1, pCAL2 e pCAL3. As pró-catepsinas L recombinantes foram purificadas por cromatografia de afinidade e a incubação em pH ácido resultou na formação das enzimas maduras CAL1, CAL2 e CAL3 com atividade sobre o substrato Z-FR-MCA. O anticorpo policlonal anti-pCAL2 foi produzido em coelho e reconheceu pCAL2 e CAL2 em immunoblots. Experimentos de immunoblots com diferentes tecidos de T. molitor mostraram que o anticorpo policlonal anti-pCAL3 reconheceu pCAL3 e CAL3 nos dois terços anteriores do intestino médio de larvas de T. molitor. Estudos de imunocitolocalização indicam que a catepsina L 3 ocorre em vesículas no intestino médio anterior e em microvilosidades no intestino médio posterior. Para os experimentos de cristalização, nós expressamos pCAL1, pCAL2 e pCAL3 como mutantes Cys25→Ser inativos. pCAL3Cys26Ser foi cristalizada por difusão de vapor (gota sentada) contra 0,1-1,6M de dihidrogênio fosfato de amônio. Os cristais são monoclínicos com grupo espacial C2 e parâmetros de célula: a=57,634 Å, b=89,322 Å, c=70,076 Å, α=γ=90°, β=92,502° e uma molécula na unidade assimétrica. A e strutura foi determinada por substituição molecular usando a estrutura de Fasciola hepatica (42% de identidade) como modelo. O modelo foi refinado a 2,1 Å com fator R final de 16,19% (Rfree=20,5%). pCAL2Cys25Ser foi cristalizada por difusão de vapor (gota sentada) contra acetato de sódio 0,2M, cacodilato de sódio 0,1M pH6,6-6,7 e 20% de PEG 8000. Os cristais são triclínicos com grupo espacial P1 e parâmetros de célula: a=51,669 Å, b=52,37 Å, c=59,716 Å, α= 91,278°, γ=109,586°, β=91,547° e duas moléculas na unidade assimétrica. A estrutura foi determinada por substituição molecular usando a estrutura da pCAL3 (44% de identidade) como modelo. O modelo foi refinado a 2,0 Å com fator R final de 17,61% (Rfree=22,48%). A estrutura terciária da pró-catepsinas L digestivas é muito similar as estruturas de cisteína proteinases da família da papaína / Cathepsin L is a cysteine proteinase of the papain family (clan CA, family C1), which is the most known among the cysteine proteinases. Cathepsin L, like other proteinases of family C1, is synthesized as an inactive proenzyme that is activated by propeptide removal. The propeptide of cathepsin L-like subfamily contain a highly conserved motif, the so called ERFNIN motif. Cathepsin L corresponds to the major digestive proteinase in Tenebrio molitor. In our laboratory, 3 procathepsins L (pCALs) were cloned and sequenced from a cDNA library prepared from T. molitor larval midguts: pCAL1 (lysosomal CAL), pCAL2 and pCAL3 (digestive enzymes). These proteinases have ERFNIN motif and 3 residues directly involved in catalysis: Cys25, His169, Asn175 with Gln19 (papain numbering). In this work we report the cloning into the expression vector and bacterial expression of the sequences coding pCAL1, pCAL2 and pCAL3. The recombinant procathepsins L were purified by affinity chromatography and activation of these enzymes occurs under acidic conditions. The cathepsins L (CAL1, CAL2 and CAL3) were able to hydrolyse Z-FR-MCA. The polyclonal antibody anti-pCAL2 was produced in rabbit and recognized pCAL2 and CAL2 on immunoblots. Immunoblot analyses of different T. molitor larval tissues demonstrated that the polyclonal antibody anti-pCAL3 recognised pCAL3 and CAL3 in the anterior two-thirds of midgut tissue of T. molitor larvae. Immunolocalization studies indicate that cathepsin L 3 occurs in vesicles in the anterior midgut and microvilli in posterior midgut. To crystallographic studies we expressed pCAL1, pCAL2 and pCAL3 as inactive Cys25→Ser mutants. pCAL3Cys26Ser was crystallized by vapor diffusion in sitting drops against 0.1-1.6 M mono-ammonium dihydrogen phosphate. The crystals are monoclinic, belonging to space group C2, with cell parameters: a = 57.634 Å, b = 89.322 Å, c = 70.076 Å, α = γ =90°, β = 92.502° and contain one molecule in the asymmetric unit. The structure was determined by molecular replacement using the structure of Fasciola hepatica procathepsin L (42.5% identity) as a model. The model was refined at 2.1 Å resolution with an R factor of 16.19% (Rfree = 20.5%). pCAL2Cys25Ser was crystallized by vapor diffusion in sitting drops against 0.2M sodium acetate, 0.1M sodium cacodylate pH 6.6-6.7 and 20% polyethylene glycol 8,000. The crystals are triclinic, belonging to space group P1, with cell parameters: a = 51.669 Å, b = 52.37 Å, c = 59.716 Å, α = 91.278° γ = 109.586°, β = 91.547° and contain two molecules in the asymmetric unit. The structure was determined by molecular replacement using the structure of procathepsin L 3 (44 % identity) as a model. The model was refined at 2.0 Å resolution with an R factor of 17.61% (Rfree = 22.48%). The tertiary structure ofdigestive procathepsins L is very similar to papain-like cysteine proteinases structures

Dermicidina (DCD) é um gene mapeado no cromossomo 12, lócus 12q13.1, e codifica uma proteína de 110 aminoácidos, que sofre um processamento proteolítico, gerando peptídeos ativos. O peptídeo C-terminal (DCD-1L) de 48 aminoácidos tem uma carga -2, e exerce função antibacteriana e antifúngica, e o peptídeo C-terminal splice variante, denominado DCD-SV de 59 aminoácidos, tem carga neutra, e suas propriedades ainda não foram estabelecidas. Neste trabalho são apresentados os resultados da expressão, purificação e sequenciamento da DCD nativa produzida em E. coli BL21 transformada com o vetor pAE-DCD. Na segunda parte são descritas as análises físico-químicas e bioquímicas da interação dos peptídeos sintéticos DCD-1L e DCD-SV com vesículas lipídicas gigantes e vesículas unilamelar grandes sintetizadas com palmitoil-oleoil-fosfatidilcolina. As preferenciais estruturais dos peptídeos foram investigadas por espectroscopia de Dicroísmo Circular. Nossos resultados sugerem que a DCD-SV tem alta propensão para adotar uma estrutura helicoidal permitindo sua inserção e oligomerização em membranas biomiméticas, e possível formação de canais de condutância molecular. / Dermicidin (DCD) is mapped a gene on chromosome 12, locus 12q1.13 whose 110 amino acids protein is proteolytically processed to N and C-terminal peptides. The 48-amino acid C-terminal peptide (DCD-1L) has -2 net charges and display antibacterial and antifungal properties and the 59-amino acid splice variant C-terminal peptide (DCD-SV) has neutral net charge; however, its structure and biological function are unknown. Here we show the results of expression, purification and amino acid sequencing of recombinant DCD protein produced in E.coli transformed with pAE-DCD vector. We also describe the results of physical-chemical and biochemical analyses showing the visible differences between the interactions of DCD-1LL and DCD-SV synthetic peptides with giant unilamellar vesicles and large unilamellar vesciles made of palmitoyl-oleoyl phosphatidylcholine, used as biomimetic membranes. The structural preferences of peptides were analyzed by circular dichroism spectroscopy. Our results suggest that DCD-SV peptide has higher propensity to adopt helicoidal structure enabling it to insert into mimetic membranes, undergo oligomerization and formation of conductance channel.

A troponina C é o componente do complexo heterotrimérico troponina ao qual o Ca2+ se associa. Essa associação torna a contração muscular um processo regulado por Ca2+. Pearlstone et al. (Biochemistry 31, 6545, (1992)) utilizaram o cDNA da troponina C de músculo esquelético de galinha (sTnC) para construir um mutante onde a fenilalanina da posição 29 foi substituída por triptofano (mutante F29W). Esse mutante permitiu que a ligação de Ca2+ aos sítios regulatórios amino terminais fosse acompanhada através de técnicas fluorescentes. Entretanto, algumas propriedades da sTnC foram alteradas pela mutação (Li et al. (1995) Biochemistry 34, 8330). No presente estudo, ensaios de ligação direta de Ca2+ bem como titulações de Ca2+ seguidas por fluorescência foram usadas para melhor se entenderem os efeitos da mutação Phe→Trp na posição 29 bem como a influência de certos aminoácidos componentes do sítio de ligação de Ca2+ nas propriedades do domínio regulatório. Dois novos mutantes foram construídos nos quais os análogos do triptofano 5-hidroxitriptofano ou 7 -azatriptofano foram introduzidos na posição 29 (resultando nos mutantes F29HW e F29ZW, respectivamente). Os dados mostraram que, quando comparada com a sTnC, a afinidade por Ca2+ dos sítios amino terminais foi elevada na F29W em aproximadamente seis vezes, três vezes na F29ZW e levemente diminuída na F29HW . A curva de fluorescência associada à ligação de Ca2+ à F29ZW mostrou-se bimodal, com cada fase podendo ser relacionada à ligação de Ca2+ a cada um dos sítios regulatórios. Esta é a primeira descrição através de técnicas de fluorescência da ligação sequencial de Ca2+ aos sítios amino terminais. Para investigar a influência de cada um dos sítios amino terminais nas propriedades de ligação ao Ca2+ ou propriedades fluorescentes da sTnC, F29W, F29HW e F29ZW , construímos mutantes duplos e triplos através da substituição do aspartato da posição 30 ou 66 (ou ambos) por alanina. Essas mutações afetam respectivamente a capacidade de ligação ao Ca2+ dos sítios I e II. Os dados mostraram que: 1) nas concentrações de Ca2+ analisadas, a mutação Asp→Ala na posição 30 impede somente a ligação de Ca2+ ao sítio I, enquanto a mutação Asp → Ala na posição 66 impede a ligação de Ca2+ aos sítios I e II, e 2) a mutação Asp → Ala na posição 30 torna silenciosa a substituição da fenilalanina da posição 29 por Trp, 5-hidroxitriptofano ou 7-azatriptofano. Concluímos que o sítio I é essencialmente inativo sem a ligação prévia de Ca2+ ao sítio II e que a posição 29 influencia a afinidade ao Ca2+ do sítio I \"ajustado\". / Troponin C is the Ca2+ binding component of heterotrimeric troponin. It makes skeletal muscle contraction a Ca2+ regulated process. We have previously used the cDNA of chicken skeletal TnC (sTnC) to construct a mutant where phenylalanine at position 29 was replaced by tryptophan (F29W mutant) [Pearlstone et al. (1992) Biochemistry 31, 6545]. This mutant allowed calcium binding to the regulatory amino terminal sites to be followed through fluorescence techniques, but altered some properties of sTnC [Li et al. (1995) Biochemistry 34, 8330]. In the present study, direct calcium binding assays and fluorescence followed calcium titrations were used to better understand the effects of the Phe→Trp mutation at position 29 as well as the influence of each amino site on the calcium binding properties of the regulatory domain. Two new mutants were constructed in which the tryptophan analogs 5-hydroxytryptophan or 7-azatryptophan were introduced at position 29 (resulting in F29HW and F29ZW mutants, respectively). The data showed that when compared to sTnC, the Ca2+ affinity of amino sites was increased sixfold in F29W, nearly threefold in F29ZW and slightly decreased in F29HW. The F29ZW fluorescence followed Ca2+ titration displayed a bimodal curve that could be related to Ca2+ binding to each of the amino sites. This is the first report of fluorescence detection of the sequential Ca2+ binding to the regulatory sites. To investigate the influence of each amino site in the calcium binding or fluorescence properties of sTnC, F29W, F29HW and F29ZW, we have constructed double and triple mutants by replacing aspartate at position 30 or 66 (or both) by alanine. These mutations affect respectively the binding capacity of sites I and II. The data showed that: 1) in the calcium concentration range analyzed, the Asp→Ala mutation at position 30 impairs calcium binding to site I on1y, while Asp→Ala mutation at position 66 impairs calcium binding to both sites I and II, and 2) the Asp→Ala mutation at position 30 makes silent the replacement of Phe at position 29 by Trp, 5-hydroxytryptophan or 7-azatryptophan. We conclude that site I is essentially defunct without previous binding to site II and that position 29 influences the affinity of this adjusted site I.

Um dos maiores desafios atuais na biologia estrutural é como lidar com flexibilidade de proteínas. Além do desafio experimental, uma limitação teórica é a falta de uma linguagem matemática conveniente para representação do espaço conformacional de proteínas. As representações mais populares apresentam diversas limitações, que se refletem nas dificuldades associadas à análise de ensembles conformacionais. Nesse contexto, a aplicação de geometria diferencial (GD) e teoria da informação (TI) foi pouco explorada. Neste trabalho investigamos o uso de descritores de GD e TI como uma representação matemática do espaço conformacional de proteínas aplicada à análise de ensembles conformacionais. O cálculo dos descritores de GD consiste em representar o backbone de proteínas como curvas espaciais e caracterizá-las utilizando os seus valores de curvatura, κ, e torção, τ . Baseado nesses valores, definimos medidas de flexibilidade, de distância entre conformações e aplicamos uma estratégia de clustering para identificação de estados conformacionais. Para permitir a aplicação de TI, desenvolvemos um sistema de codificação desses descritores para expressar cada conformação por uma sequência de símbolos finitos. A partir dessas sequências, definimos uma medida da informação associada a um resíduo, Rres, e a uma conformação, Rconf. Para investigar sua eficácia, aplicamos os métodos propostos aos ensembles conformacionais de três sistemas testes: 1) Ubiquitina, 2) E1-DBD do HPV18 e 3) as etapas de formação do complexo c-Myb-KIX. A análise da representação por geometria diferencial se mostrou igualmente eficaz ou superior aos métodos comumente utilizados em todos os sistemas analisados. O método é especialmente útil para monitoramento de estabilidade de hélices e para análise de proteínas e regiões muito flexíveis, pois evita a necessidade de sobreposição estrutural. Os valores de Rconf se apresentaram úteis para análise de processos de enovelamento e resíduos próximos a regiões funcionais tendem a apresentar maiores valores Rres. No entanto, o papel desses resíduos é incerto e mais estudos são necessários para determinar se há e qual é seu real significado. Apesar disso, as medidas de informação se mostraram úteis para comparação de estados conformacionais e permitem levantar hipóteses testáveis em laboratório. Por fim, a representação por GD é computacionalmente conveniente, intuitiva, evita todas as limitações dos métodos popularmente utilizados e se mostrou eficaz para análise de ensembles conformacionais. / One of the major challenges of modern structural biology is how to deal with protein flexibility. Besides the experimental difficulties, a relatively overlooked theoretical challenge is the lack of a proper mathematical language to represent proteín conformational space. The most popular representations have severe limitations, which reflects on the difficulties associated with conformational ensemble analyses. However, differential geometry (GD) and information theory (TI) can help to overcome such difficulties and were not well explored in this context. Here we investigate the usage of DG and TI as a mathematical representation of protein conformational space applied to the analyses of conformational ensembles. The DG descriptors calculation consists of representing protein backbone as a spatial curve and describes it by its curvature, κ, and torsion, τ . Based on those values, the distance between conformation and flexibility measurements were defined and a clustering algorithm was applied to identify conformational states. For the application of TI, a coding system for DG descriptors was developed to express each conformation as a sequence of finite symbols. Based on those sequences, information measurements associated to a residue, Rres, and to a conformation, Rconf , were defined. To investigate its efficacy, the proposed method was applied to conformation ensembles of three test systems: 1) Ubiquitin, 2) E1-DBD of HPV18 and 3) the steps of c-Myb-KIX binding. The DG analyses show equally good or superior performance when compared with popular methods on all tested system. In addition, the methods are especially useful to monitoring helix stability and analyses of very flexible proteins (or regions), since avoids the necessity of superposing structures. The values of Rconf are useful to compare different steps of a folding process and residues near regions involved in binding events tend to present higher values of Rres. However, those residues importance is uncertain and further studies are necessary to determinate if and how those can contribute to protein function. Nevertheless, the information measurements were informative on the comparison of compare conformational states and allow to formulate a testable hypothesis. On the other hand, the GD representation is computationally convenient, intuitive and avoid most of the limitations of the popular method applied to conformational ensemble analyses.

A demanda criada pelos estudos biológicos resultou para predição da estrutura terciária de proteínas ser uma alternativa, uma vez que menos de 1% das sequências conhecidas possuem sua estrutura terciária determinada experimentalmente. As predições Ab initio foca nas funções baseadas da física, a qual se trata apenas das informações providas pela sequência primária. Por consequência, um espaço de busca com muitos mínimos locais ótimos deve ser pesquisado. Este cenário complexo evidencia uma carência de algoritmos eficientes para este espaço, tornando-se assim o principal obstáculo para este tipo de predição. A optimização Multi-Objetiva, principalmente os Algoritmos Evolutivos, vem sendo aplicados na predição da estrutura terciária já que na mesma se envolve um compromisso entre os objetivos. Este trabalho apresenta o framework ProtPred-PEO-GROMACS, ou simplesmente 3PG, que não somente faz predições com a mesma acurácia encontrada na literatura, mas também, permite investigar a predição por meio da manipulação de combinações de objetivos, tanto no aspecto energético quanto no estrutural. Além disso, o 3PG facilita a implementação de novas opções, métodos de análises e também novos algoritmos evolutivos. A fim de salientar a capacidade do 3PG, foi então discorrida uma comparação entre os algoritmos NSGA-II e SPEA2 aplicados na predição Ab initio da estrutura terciária de proteínas em seis combinações de objetivos. Ademais, o uso da técnica de refinamento por Dinâmica Molecular é avaliado. Os resultados foram adequados quando comparado com outras técnicas de predições: Algoritmos Evolutivo Multi-Objetivo, Replica Exchange Molecular Dynamics, PEP-FOLD e Folding@Home. / The demand created by biological studies resulted the structure prediction as an alternative, since less than 1% of the known protein primary sequences have their 3D structure experimentally determined. Ab initio predictions focus on physics-based functions, which regard only information about the primary sequence. As a consequence, a search space with several local optima must be sampled, leading to insucient sampling of this space, which is the main hindrance towards better predictions. Multi-Objective Optimization approaches, particularly the Evolutionary Algorithms, have been applied in protein structure prediction as it involves a compromise among conicting objectives. In this paper we present the ProtPred-PEO-GROMACS framework, or 3PG, which can not only make protein structure predictions with the same accuracy standards as those found in the literature, but also allows the study of protein structures by handling several energetic and structural objective combinations. Moreover, the 3PG framework facilitates the fast implementation of new objective options, method analysis and even new evolutionary algorithms. In this study, we perform a comparison between the NSGA-II and SPEA2 algorithms applied on six dierent combinations of objectives to the protein structure. Besides, the use of Molecular Dynamics simulations as a renement technique is assessed. The results were suitable when comparated with other prediction methodologies, such as: Multi-Objective Evolutionary Algorithms, Replica Exchange Molecular Dynamics, PEP-FOLD and Folding@Home.

Amyloidogenic neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are an important health issue. However, the underlying molecular mechanisms of the disease-related protein aggregates, that are present in humans, are only understood partially. I have used and developed biophysical methods to study the structural and biological properties of individual aggregates of Amyloid β peptide and α-Synuclein, proteins whose aggregation is associated with the development of Alzheimer's and Parkinson's disease respectively. I expanded the single aggregate visualisation through enhancement (SAVE) technique, which is a method based on the fluorescent dye Thioflavin T (ThT) that reversibly bind to the aggregates and whose fluorescence increases upon binding. I firstly explored the use of other dyes for these experiments and found that a ThT dimer has higher affinity to α-Synuclein aggregates in vitro. I then applied the SAVE method to the cerebral spinal fluid (CSF) of a cohort of AD patients and control CSF and observed no clear difference in aggregate number. However, these experiments provided insights into how antibodies bind the aggregates in human CSF. I could show, that despite altering the Ca2+ influx into both cells and vesicles, the antibody did not measurably affect the aggregate structure. To study the size specific effects of the Amyloid β 42 (Aβ42) peptide in more detail, I used and optimised gradient ultracentrifugation combined with single aggregate imaging to study the structural properties of the isolated aggregates. This aggregation kinetic independent method allowed me to compare the properties of fluorescently labelled and unlabelled Aβ42 and characterize the size dependent properties of aggregates in a single experiment. Since I could measure the relative concentration of different size aggregates it was also possible to compare the properties of single aggregates of different sizes. I then used biological assays to examine the ability of aggregates to permeabilise membranes resulting in the entry of calcium ions, and their ability to induce TNFα production in microglia cells. Both processes are thought to play key roles in the development of AD. I found that small soluble oligomers are most potent at inducing Ca2+ influx, whereas longer protofilaments are the most potent inducers of TNFα production. My results suggest that the mechanism by which aggregates damage cells changes as aggregation proceeds, as longer aggregates with different structures are formed. Protofilaments with a diameter of 1 nm or less have a structure that could make them particularly potent at causing the signalling of toll-like receptors, providing a molecular basis for their ability to induce TNFα production.

ATP-dependent chromatin remodelers have been proposed to act sequentially, and to a certain extent non-redundantly, in the priming stages of the DNA Damage Response pathways by establishing chromatin in lesion sites ready to act as a scaffold for repair factors or to be displaced in order to allow DNA repair. Among remodeling factors proposed to play a role in DNA repair is SMARCAD1, a poorly characterized, non-canonical member of the SWR1-like family of SNF2/SWI2 superfamily of ATPases, which has recently been identified as a potential target for ATM/ATR phosphorylation at canonical and non-canonical sites upon DNA damage. The actual mechanism for SMARCAD1 recruitment and involvement in DNA remodeling is still unknown, and unlike most other chromatin remodelers, SMARCAD1 does not contain DNA- or histone-binding domains frequently accompanying such proteins. Instead, in addition to the core ATPase domain, only two CUE domains (a type of helical ubiquitin-binding domain) have been identified. This thesis presents the findings of an investigation intended to structurally characterize SMARCAD1 by dissecting and identifying its domain architecture, and examining the activity and ligand selectivity of its binding domains in the functional context of DNA damage repair. The solution NMR structure of the CUE1 domain is presented, describing a triple helix bundle consistent with other members of the family. Furthermore, a novel SUMO interacting motif was identified and through a combination of NMR titrations and phospho-proteomics analysis, shown to be constitutively phosphorylated which excludes the possibility of DNA damage dependent ATM targeting as the recruitment mechanism for DNA repair. Additionally, it is demonstrated that both CUE domains are poor binders of mono-ubiquitin, however CUE1 specifically mediates the high affinity binary interaction with the transcriptionally repressive master regulator KAP1. This interaction was shown to be independent of post-translational ubiquitylation but rather sustained through direct interaction with the dimeric RBCC domain of KAP1. Finally, mass spectrometry profiling of domain-dependent interactions (based on differential abundance relative to changes due to chemically induced DNA damage) suggests SMARCAD1 may be involved in p53 transcriptional regulation through interactions maintained with CUE1 prior to DNA damage, whereas the SIM domain selectively targets protein interactions upon DNA damage that simultaneously activate p53 transcriptional control and recruit SMARCAD1 to DNA damage repair pathways.