Studien zur Proteintranslokation in Escherichia coli : Untersuchung der Membranproteine SecYEG und YidC unter Verwendung biochemischer und kristallographischer Methoden

Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.Der Transport von Proteinen in zelluläre Membranen hinein oder durch diese hindurch wird durch die konservierte und überall anzutreffende Sec-Maschinerie vermittelt. Das Sec-Homolog in der inneren Membran von Escherichia coli ist SecYEG. Der Sec-vermittelte Einbau von vielen Membranproteinen wird unterstützt von YidC, einem weiteren Protein in der inneren Membran von Escherichia coli. YidC führt zusätzlich den Einbau einiger Proteine Sec-unabhängig durch. YidC gehört zu einer konservierten aber strukturell uncharakterisierten Familie von Proteinen, die wichtig für die Biogenese von Membranproteinen sind und hat Homologe in Mitochondrien und Chloroplasten. Durch Modifikation eines früheren Kristallisations-Protokolles wurde zweidimensionale Kristalle von SecYEG in Gegenwart des Signalsequenz-Peptides von LamB gezüchtet. Das Aufnehmen von Strukturdaten mittels Elektronen-Kryomikroskopie und die Berechnung einer Differenzstruktur, welche eine frühere SecYEG-Projektionsstruktur mit der von SecYEG kristallisiert in Gegenwart von Substrat vergleicht, demonstrierte eine Reihe neuer bzw. fehlender Dichten. Diese deuten auf das Binden von Signalpeptid in räumlicher Nähe zur Translokationspore sowie auf signifikante Umlagerungen in der Nähe des lateralen Austrittsortes für Transmembrandomänen in SecYEG hin. Die Differenzstruktur legt nahe, dass dimeres SecYEG ein asymmetrisches Molekül ist, bestehend aus einem aktiven und einem inaktiven SecYEG-Monomer. Detergenzentfernung aus einem Gemisch von gereinigtem YidC und Lipiden brachte zweidimensionale Kristalle hervor, welche bezüglich ihrer Bildung sehr abhängig von Ionenstärke und Lipidkomposition waren. Eine elektronen-kryomikroskopische Untersuchung der in hydratisiertem Zustand gefrorenen Kristalle und Bildverarbeitung machten strukturelle Details mit einer Auflösung von etwa 10 Å sichtbar. Mitteln von Einzelbildern zu zwei alternativen Projektionsstrukturen in p2 bzw. p121_a Symmetrie brachte grundsätzlich gleiche strukturelle Merkmale hervor. Vier YidC-Monomere bilden eine Einheitszelle (Dimensionen 82 x 71 Å, eingeschlossener Winkel 85 ° bzw. 90 °) und scheinen als zwei Dimere vorzuliegen, welche in gegensätzlicher Orientierung in die Membran eingebettet sind. Ein Bereich geringer Dichte im Zentrum eines jeden YidC-Monomers stellt möglicherweise eine Einstülpung der Membran dar, die besondere Bedeutung für den Einbau von Substratproteinen in die Lipid-Doppelschicht haben könnte

Nicotinic acetylcholine receptors and their interactions with allosteric ligands

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels (pLGICs) expressed widely throughout the body, including in the peripheral nervous system, central nervous system and at the neuromuscular junction. nAChRs are of therapeutic interest due to their involvement in several pathophysiological conditions. The most widely expressed nAChR subtypes, α7 and α4β2 have attracted a lot of attention and many allosteric ligands have been pharmacologically and chemically characterised for these receptors. However, much remains to be understood about where and how these ligands bind to the receptors and modulate their function. This thesis has focussed on a set of transmembrane binding allosteric modulators for the α7 nAChR and sought to aid understanding of their interactions with their target receptor by building models of nAChRs in physiologically relevant states. A transmembrane error in the only example of a pLGIC structure determined in a native lipid membrane environment, the T. marmorata nAChR, has been corrected through modelling and refinement into previously determined electron cryo-microscopy density maps, in putative closed and open conformations. The refined models offer important reference structures for anyone working in the pLGIC field and here have been used as templates to model the α7 nAChR. A consensus docking protocol has been developed and was utilised in conjunction with the α7 models to predict binding modes for a set of allosteric modulators and provide insight into how they may elicit distinct pharmacology. Based on binding modes of allosteric modulators predicted by the consensus docking protocol, pharmacophores were generated for use in ligand-based virtual screening and allosteric modulators have been uncovered for α7 and α4β2 nAChRs from the existing pharmacopeia. Further to this, novel reactive chemical probes have been developed and synthesised to study the covalent incorporation of allosteric modulators into nAChRs

Developing models to study the mechanisms of weakness and myotonia in Periodic Paralysis

Periodic paralysis (PP) is a disorder characterised by episodic attacks of paralysis, caused by mutations of skeletal muscle voltage gated ion channels. Although episodes eventually subside, patients develop progressive muscle weakness and frequently, myopathy. The relationship between this progression and the associated mutations is not understood. I propose that the longer term defect might result from disordered calcium signalling secondary to altered excitability, and its impact on mitochondrial function. I sought models where these aspects of muscle signalling could be studied. These were: A genetic model derived from patients: patient derived fibroblasts were virally transduced with MyoD to generate myoblasts, which were differentiated into myotubes with patient specific gene mutations. A pharmacological model: generated by treating neonatal rat myotube cultures with barium (an inhibitor of potassium channels) and low extracellular potassium to simulate attacks of PP. Treated cultures displayed more frequent spontaneous calcium fluctuations. Mitochondrial membrane potential was not affected by the treatment, but expression of TFAM (mitochondrial transcription factor A; a regulator of mitochondrial transcription and biogenesis) was upregulated, suggesting activation of retrograde signalling pathways. A mouse model: collaborators at MRC Harwell generated mice carrying a mutation (c.1744A>G; p.Ile582Val) equivalent to a novel point mutation in SCN4A, one of the ion channel genes associated with PP. Measurements in vivo established that affected mice show muscle weakness and delayed fatigue during tetanic responses. Calcium handling and mitochondrial function were analysed in single isolated myofibres. Calcium handling was not affected, however mitochondrial membrane potential was reduced in fibres from the PP mice and distribution was also affected, with fewer intermyofibrillar mitochondria, indicating altered mitochondrial bioenergetics. Thus I describe several approaches to investigate mechanisms that cause progressive weakness and myopathy in PP, and assess the relative merits of each approach. Furthermore, results suggest that a shift toward a more oxidative phenotype is taking place

Developments on Metallomic and Proteomic strategies for early diagnosis of Alzheimer´s Disease

The current Thesis deals with the searching of biomarkers for an early Alzheimer’s disease diagnosis. The two first chapters have been devoted to the optimization of methods for the determination of several elements by ICP-MS (use of dried blood spot, DBS; and discrete sampling followed to introduction in ICP-MS). In the second part of this thesis regarding proteomics, several proteins for differentiating three groups of population (healthy people, AD patients, and MCI patients) have been found. In a final chapter, LA conditions have been optimized for the determination of metals in the isoforms of two proteins [Serotransferrin (TRFE) and Keratin type II cytoskeletal 1 (K2C1)] after 2D electrophoresis

Use of Transmission Electron Microscopy to Identify Nanocrystals of Challenging Protein Targets

Existing crystallographic methods require micro-meter sized crystals for structure determination. However, due to their intrinsic disorder, many biological molecules are not amenable to the formation of such large crystals. X-ray free electron lasers (X-FEL) provide a new avenue to determine crystal structures of such challenging biological targets, as this technology enables the acquisition of diffraction patterns from nano-meter sized crystals. As proof of principle, X-FELs have been employed to determine crystal structures obtained using conventional crystallographic methods. A major limitation to the application of X-FEL technology to de novo structure determination of challenging targets is the inability to identify protein nanocrystals (NCs) that are suitable for diffraction by X-FEL. Here, I establish a method using Transmission Electron Microscopy (TEM) to reliably identify and characterize protein NCs. I then use this method to compare crystallographic seeding techniques to identify conditions that yield larger and higher quality NCs. Negative stain Transmission Electron Microscopy (TEM) was used to detect NCs of several challenging targets. One such challenging target is a Family B G-Protein Coupled Receptor, the parathyroid hormone receptor (PTHR1). Crystallization trials of the PTHR1 were performed to isolate potential NC containing drops. UV tryptophan fluorescence microscopy was employed to identify UV positive granular aggregates. Positive samples were examined by negative stain TEM with the aim of distinguishing NCs from protein aggregates. In those PTHR1 NCs that were identified, the presence of negative stained lattices was used as an indicator of their proteic nature. This method was successfully employed to identify protein NCs of seven additional challenging targets. Crystal quality was determined by calculating fast Fourier transforms of NC lattices. TEM was also used to visualize crystallographic seeds for the first time, and identify optimal seeding conditions. The quality of seeds produced by traditional seeding methods were compared to those produced by “nanoseeding,” a novel method in which multiple millimeter seed beads are used. The nanoseeding approach proved to be far superior to traditional methods at producing larger and higher quality seeds. This work demonstrates that TEM identified NCs, as well as fragmented NCs are powerful tools for optimizing and growing crystals

New insights into mutable collagenous tissues: an inspiring model for tissue regeneration

Tese de mestrado integrado. Engenharia Biomédica. Universidade do Porto. Faculdade de Engenharia. 201

Unraveling the intricacies of spatial organization of the ErbB receptors and downstream signaling pathways

Faced with the complexity of diseases such as cancer which has 1012 mutations, altering gene expression, and disrupting regulatory networks, there has been a paradigm shift in the biological sciences and what has emerged is a much more quantitative field of biology. Mathematical modeling can aid in biological discovery with the development of predictive models that provide future direction for experimentalist. In this work, I have contributed to the development of novel computational approaches which explore mechanisms of receptor aggregation and predict the effects of downstream signaling. The coupled spatial non-spatial simulation algorithm, CSNSA is a tool that I took part in developing, which implements a spatial kinetic Monte Carlo for capturing receptor interactions on the cell membrane with Gillespies stochastic simulation algorithm, SSA, for temporal cytosolic interactions. Using this framework we determine that receptor clustering significantly enhances downstream signaling. In the next study the goal was to understand mechanisms of clustering. Cytoskeletal interactions with mobile proteins are known to hinder diffusion. Using a Monte Carlo approach we simulate these interactions, determining at what cytoskeletal distribution and receptor concentration optimal clustering occurs and when it is inhibited. We investigate oligomerization induced trapping to determine mechanisms of clustering, and our results show that the cytoskeletal interactions lead to receptor clustering. After exploring the mechanisms of clustering we determine how receptor aggregation effects downstream signaling. We further proceed by implementing the adaptively coarse grained Monte Carlo, ACGMC to determine if \u27receptor-sharing\u27 occurs when receptors are clustered. In our proposed \u27receptor-sharing\u27 mechanism a cytosolic species binds with a receptor then disassociates and rebinds a neighboring receptor. We tested our hypothesis using a novel computational approach, the ACGMC, an algorithm which enables the spatial temporal evolution of the system in three dimensions by using a coarse graining approach. In this framework we are modeling EGFR reaction-diffusion events on the plasma membrane while capturing the spatial-temporal dynamics of proteins in the cytosol. From this framework we observe \u27receptor-sharing\u27 which may be an important mechanism in the regulation and overall efficiency of signal transduction. In summary, I have helped to develop predictive computational tools that take systems biology in a new direction.\u2

Atomic force microscopy to elucidate lipid membranes enhanced by engineered liposomes

[eng] The research in this thesis aimed to study and generate an engineered formulation that can fuse with cell membranes and carry drugs or other compounds into cells. HeLa cells were chosen as the target cells and prior to their use, a model membrane mimicking the lipid membrane of HeLa cells was developed. Starting from the basic components and using a bottom-up approach, different phospholipids were studied and compared to identify the construction blocks of liposomes and assess the effects of cholesterol on these phospholipids. After selecting the desired composition, a membrane model mimicking the HeLa cell membrane was developed to test its fusion with the engineered liposomes and to understand the fusion process before starting in vitro assays with living HeLa cells. In the in vitro assays, the engineered liposomes were able to fuse with the cell membrane as well as carry and liberate a model drug (methotrexate) into the cells, demonstrating that the engineered liposomes can work efficiently as nanocarriers. Across the entire thesis, one technique was constantly used, atomic force microscopy (AFM). This technique enables the study of the smallest samples, such as lipid monolayers, as well as larger samples, like HeLa cells. AFM can also be used to obtain the physicochemical properties of samples using the force spectroscopy mode, allowing the analysis of samples and providing insight into the nanomechanics of the samples studied. Several techniques were used in this thesis, including the application of a Langmuir-Blodgett trough to study the physicochemical properties of lipids, fluorescence resonance energy transfer (FRET) to determine the fusion of the engineered liposomes, visualization techniques like AFM and confocal microscopy, as well as viability assays to test the toxicity of the engineered liposomes to HeLa cells. Finally, we demonstrated the ability of the engineered liposomes to fuse with cells, acting as nanocarriers based on their physicochemical properties. The ability of the membrane model to mimic the HeLa cell lipid membrane was also validated.[cat] Aquesta tesi té com a objectiu l'estudi i el disseny d'una formulació capaç de fusionar i transportar fàrmacs o altres molècules a les cèl·lules. Per a l'estudi, les cèl·lules objectiu seleccionades han estat cèl·lules HeLa i abans del seu ús, s'ha desenvolupat un model de membrana que imita la membrana lipídica de les cèl·lules HeLa. Partint dels components bàsics, des d'un punt de vista del desenvolupament “bottom-up”, s'han estudiat i comparat diferents fosfolípids per trobar els blocs de construcció adequats per als liposomes, també s'han estudiat els efectes del colesterol sobre aquests fosfolípids. Després de seleccionar la composició desitjada, s'ha desenvolupat una formulació que imita en composició la membrana cel·lular de les cèl·lules HeLa per provar la fusió dels liposomes dissenyats i per intentar entendre el procés de fusió abans d'iniciar els assajos in vitro amb cèl·lules HeLa. Pel que fa als assajos in vitro, els liposomes han demostrat ser capaços de fusionar-se a la membrana, així com transportar i alliberar un fàrmac model (metotrexat) a les cèl·lules, demostrant que els liposomes dissenyats en aquesta tesi són capaços de funcionar de manera eficient com a “nanocarriers”. Al llarg d’aquesta tesi, una tècnica ha estat constantment present, la microscòpia de força atòmica (AFM), ja que ofereix la possibilitat de realitzar estudis des de les mostres més petites, com l'estudi de monocapes lipídiques, fins a mostres més grans com les cèl·lules HeLa. Aquesta tècnica també permet fer observacions fisicoquímiques de qualsevol d'aquestes mostres mitjançant el mode d'espectroscòpia de força que permet sondejar les mostres i obtenir informació sobre la nanomecànica de les mostres estudiades. Amb aquesta finalitat s'han utilitzat diverses tècniques, tant les que han ajudat a estudiar les propietats fisicoquímiques dels lípids, com el de Langmuir-Blodgett, com altres per determinar els efectes de fusió dels liposomes com la transferència d'energia per ressonància fluorescent (FRET) o tècniques de visualització com l’AFM o microscòpia confocal i fins i tot tècniques de viabilitat per provar la viabilitat de la formulació a les cèl·lules HeLa. Finalment, hem desenvolupat i demostrat les capacitats dels liposomes per fusionar-se amb les cèl·lules, podent, en funció de les seves propietats fisicoquímiques, actuar com a “nanocarriers”. El model de membrana que imita les cèl·lules HeLa s'ha validat corroborant la capacitat per imitar la membrana lipídica de les cèl·lules HeLa reals