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Single Molecule Study of Beta-Carotene using Scanning Tunneling Microscope (Up-close and Personal Investigation of Beta-Carotene)Skeini, Timur 05 August 2010 (has links)
No description available.
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STM studies of charge transfer and transport through metal-molecule complexes on ultrathin insulating filmsChoi, Taeyoung 21 March 2011 (has links)
No description available.
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The Formation of Two Dimensional Supramolecular Structures and Their Use in Studying Charge Transport at the Single Molecule Level at the Liquid-Solid InterfaceAfsari Mamaghani, Sepideh January 2015 (has links)
Understanding charge transport through molecular junctions and factors affecting the conductivity at the single molecule level is the first step in designing functional electronic devices using individual molecules. A variety of methods have been developed to fabricate metal-molecule-metal junctions in order to evaluate Single Molecule Conductance (SMC). Single molecule junctions usually are formed by wiring a molecule between two metal electrodes via anchoring groups that provide efficient electronic coupling and bind the organic molecular backbone to the metal electrodes. We demonstrated a novel strategy to fabricate single molecule junctions by employing the stabilization provided by the long range ordered structure of the molecules on the surface. The templates formed by the ordered molecular adlayer immobilize the molecule on the electrode surface and facilitate conductance measurements of single molecule junctions with controlled molecular orientation. This strategy enables the construction of orientation-controlled single molecule junctions, with molecules lacking proper anchoring groups that cannot be formed via conventional SMC methods. Utilizing Scanning Tunneling Microscopy (STM) imaging and STM break junction (STM-BJ) techniques combined, we employed the molecular assembly of mesitylene to create highly conductive molecular junctions with controlled orientation of benzene ring perpendicular to the STM tip as the electrode. The long range ordered structure of mesitylene molecules imaged using STM, supports the hypothesis that mesitylene is initially adsorbed on the Au(111) with the benzene ring lying flat on the surface and perpendicular to the Au tip. Thus, long range ordered structure of mesitylene facilitates formation of Au-π-Au junctions. Mesitylene molecules do not have standard anchoring groups providing enough contact to the gold electrode and the only assumable geometry for the molecules in the junction is via direct contact between Au and the π system of the benzene ring in mesitylene. SMC measurements for Au/mesitylene/Au junctions results in a molecular conductance value around 0.125Go, two orders of magnitude higher than the measured conductance of a benzene ring connected via anchoring groups. We attributed this conductance peak to charge transport perpendicular to the benzene ring due to direct coupling between the π system and the gold electrode that happens in planar orientation. The conductance we measured for planar orientation of benzene ring is two order of magnitude larger than conductance of junctions formed with benzene derivatives with conventional linkers. Thus, altering the orientation of a single benzene-containing molecule between the two electrodes from planar orientation to the upright attached via the linkers, results in altering the conductivity in a large order. Based on these findings, by utilizing STM imaging and STM-BJ in an electrochemical environment including potential induced self-assembly formation of terephthalic acid, we designed an electrochemical single molecule switch. Terephthalic acid forms large domains of ordered structure on negatively charged Au(111) surface under negative electrochemical surface potentials with the benzene ring lying flat on the surface due to hydrogen bonding between carboxylic acid groups of neighboring molecules. Formation of long range ordered structure facilitates direct contact between the π system of the benzene ring and the gold electrodes resulting in the conductance peak. On positively charged Au(111), deprotonation of carboxylic acid groups leads to absence of long range ordered structure of molecules with planar orientation and absence of the conductance peak. In this case alternating the surface (electrode) potential from negative to positive charge densities induces a transition in the adlayer structure on the surface and switches conductance value. Hence, electrochemical surface potential can, in principle, be employed as an external stimulus to switch single molecule arrangement on the surface and the conductance in the junction. The observation of conductance switching due to molecule’s arrangement in the junction lead to the hypothesis that for any benzene derivative, an orientation-dependent conductance in the junction due to the contact geometry (i.e. electrode-anchoring groups versus direct electrode-π contact) should be expected. Conventional techniques in fabricating single molecule junctions enable accessing charge transport along only one direction, i.e., between two anchoring groups. However, molecules such as benzene derivatives are anisotropic objects and we are able to measure an orientation-dependent conductance. In order to systematically study anisotropic conductivity at single molecule level, we need to measure the conductance in different and well-controlled orientations of single molecules in the junction. We employed the same EC-STM-BJ set up for SMC measurements and utilize electrochemical potential of the substrate (electrode) as the tuning source to variate the orientation of the single molecule in the junction. We investigated single molecule conductance of the benzene rings with carboxylic acid functional groups in two orientations: one with the benzene ring bridging between two electrodes using carboxylic acids as anchoring groups (upright); and one with the molecule lying flat on the substrate perpendicular to the STM tip (planar). Physisorption of these species on the Au (111) single crystal electrode surface at negative electrochemical potentials results in an ordered structure with the benzene ring in a planar orientation. Positive electrochemical potentials cause formation of the ordered structure with molecules standing upright due to coordination of a deprotonated carboxyl groups to the electrode surface. Thus, formation of the single molecule junction and consequently conductivity measurements is facilitated in two directions for the same molecule and anisotropic conductivity can be studied. In engineering well-ordered two-dimensional (2-D) molecular structures with controlled assembly of molecular species, pH can be employed as another tuning source for the molecular structures and adsorption in experiments conducted in aqueous solutions. Based on simple chemical principles, amine (NH2) groups are hydrogen bond acceptors and donors. Amines are soluble in water and protonation results in protonated (NH3+) and unprotonated (NH2) amine groups in acidic and moderately acidic/neutral solutions, respectively. Thus, amines are suitable molecular building blocks for fabricating 2-D supramolecular structures where pH is employed as a knob to manipulate intermolecular hydrogen bonding leading to phase transitions. We investigated pH induced structural changes in the 1,3,5–triaminobenzene (TAB) monolayer and the formation/disruption of hydrogen bonds between neighboring molecules. Our STM images indicate that in the concentrated acidic solution, the protonated amine groups of TAB are not able to form H-bonds and long range ordered structure of TAB does not form on the Au(111) surface. However, in moderately acidic solution (pH ~ 5.5) at room temperature, protonation on the ring carbon atom generates species capable of forming H-bonds leading to the formation of the long range ordered structures of TAB molecules. Utilizing EC-STM set up, we investigated the controllable fabrication of a TAB 2-D supramolecular structure based on amine-amine hydrogen bonding and effect of pH in formation of ordered/disordered TAB network. / Chemistry
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From Purification to Drug Screening: CFTR TM3/4 Mutants as Models for Membrane Protein Misfolding in DiseaseSchenkel, Mathias Rolf 22 April 2024 (has links)
Membrane proteins are of undeniable importance for cell physiology across all domains of life and a loss of their function, e.g., due to mutations in their coding sequence, is almost always linked to disease. In humans, mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-gated anion channel in epithelia, give rise to cystic fibrosis (CF). Over 2100 mutations of the CFTR gene are known, however, their disease liability remains mostly undetermined. Causal therapies, i.e., small-molecule drugs that target CFTR itself, have improved the lives of people with the most common mutations (e.g. ΔF508, G551D) over the last decade. In contrast, many rare CF-phenotypic mutations are not eligible for these novel treatments and would benefit from in vitro evaluation of their molecular consequences. In vitro studies of membrane proteins are often complicated by the intrinsic hydrophobicity and aggregation susceptibility of this protein group. However, this can be avoided by using short membrane protein fragments corresponding to the smallest in vivo folding unit of the respective protein at the ER membrane. These model proteins can be easily genetically modified, expressed and purified, making them a suitable tool to pinpoint the effects of mutations.
This thesis demonstrates the utility of such a reductionist model system: TM3/4, the second helical hairpin of CFTR’s transmembrane domain 1, was used to study protein folding with a focus on disease-causing missense mutations of CFTR, which may cause CFTR misfolding in vivo. TM3/4 purification was first optimized by using a thioredoxin tag, which allowed heat purification of the fusion protein even after initial purification steps. Optimal heat treatment for maximal protein purity and recovery were determined for TM3/4 and another helical hairpin, ATP synthase subunit c. Moreover, tertiary folding of a CF-phenotypic loop mutation, E217G, introducing a non-native GXXXG interaction motif was analyzed by single-molecule Förster resonance energy transfer (smFRET) in different lipid bilayer conditions, showing unusually increased stability in comparison to wild type (WT) TM3/4. Furthermore, smFRET was used in tandem with circular dichroism and fluorescence spectroscopy to assess the effect of a specific membrane lipid, cholesterol, on TM3/4 variants showing significant changes on secondary but not tertiary structure. Lastly, a mutant library of 13 TM3/4 mutants was established to perform drug screenings with CFTR correctors – a class of small molecules rescuing or preventing misfolding of CFTR. This screening study demonstrated that (i) not all CF-phenotypic missense mutations are locally misfolded at a lipid bilayer comparable to the ER membrane; and (ii) in vitro restoration of a native WT-like conformation of locally misfolded TM3/4 mutants is not only possible but different drug-mutant pairings can be identified related to folding rescue efficiency of a given corrector on a respective mutant. The latter identified drug-mutant pairings may lead to drug repurposing if the effect can be confirmed in cell culture experiments.
In conclusion, the TM3/4 minimal model of CFTR and biophysical methods, such as smFRET, proved as versatile tools not only for investigation of mutation and lipid effects on membrane protein folding but also for drug screenings in a disease context.:1 INTRODUCTION
2 THEORETICAL BACKGROUND
2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS
2.1.1 Membrane protein families and their role in human health
2.1.2 Fundamental folding models of α-helical membrane proteins
2.1.3 Co-translational folding at the ER supported by the translocon
2.1.4 Folding-relevant interactions within membrane proteins
2.1.5 Biological membranes and lipid classes
2.1.6 Physical properties of lipid bilayers impacting membrane proteins
2.1.7 Membrane models for in vitro studies
2.2 CYSTIC FIBROSIS AND CFTR
2.2.1 Pathology of cystic fibrosis
2.2.2 Structure and function of the CFTR channel
2.2.3 A minimal model of CFTR to study rare CF mutations
2.2.4 Missense mutations within the CFTR segmental model TM3/4
2.2.5 Novel modulator therapies for the treatment of cystic fibrosis
2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING
2.3.1 Expression and purification of membrane proteins
2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding
3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS
3.1 PREAMBLE AND SUMMARY
3.2 RESULTS AND DISCUSSION
4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY
4.1 PREAMBLE AND SUMMARY
4.2 RESULTS AND DISCUSSION
5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING
5.1 PREAMBLE AND SUMMARY
5.2 RESULTS
5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol
5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor
5.2.3 Impact of Lumacaftor on membrane fluidity
5.3 DISCUSSION
6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET
6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS
6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS
7 CONCLUSIONS
8 OUTLOOK
9 MATERIALS AND METHODS
9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS
9.2 PROTEIN EXPRESSION AND PURIFICATION
9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS
9.4 SINGLE-MOLECULE FRET EXPERIMENTS
9.4.1 Labeling of TM3/4 constructs
9.4.2 Liposome preparation and reconstitution of labeled protein constructs
9.4.3 Single-molecule FRET measurements in manual mode
9.4.4 Single-molecule FRET measurements in multi-well screening mode
9.5 CIRCULAR DICHROISM SPECTROSCOPY
9.5.1 Circular dichroism to determine protein heat stability
9.5.2 Circular dichroism to study protein structure in different lipid bilayers
9.6 FLUORESCENCE SPECTROSCOPY
9.6.1 Vesicle leakage assay to test lipid bilayer stability
9.6.2 Examining lipid bilayer fluidity with fluorescent probes
10 APPENDIX
10.1 GENERATION OF A TM3/4 MUTANT LIBRARY
10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS
10.2.1 SmFRET control screenings and supporting data
10.2.2 Extracted closed state fractions from smFRET screenings
10.2.3 DLS to measure vesicle integrity after corrector addition
11 REFERENCES
12 ACKNOWLEDGEMENTS
13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG / Membranproteine sind für die Zellphysiologie aller biologischen Domänen von unbestreitbarer Bedeutung und ein Verlust ihrer Funktion, z.B. durch Mutationen in ihrer kodierenden Sequenz, ist fast immer Auslöser von Krankheiten. Beim Menschen führen Mutationen im Gen für den Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), einen ATP-abhängigen Anionenkanal in Epithelien, zu Mukoviszidose (CF). Über 2100 Mutationen des CFTR-Gens sind bekannt – ob jedoch alle Mutationen tatsächlich CF auslösen, ist weitgehend ungeklärt. Kausale Therapien, d.h. niedermolekulare Medikamente, die auf CFTR selbst abzielen, haben in den letzten zehn Jahren die Lebensqualität von Menschen mit den häufigsten Mutationen (z.B. ΔF508, G551D) verbessert. Demgegenüber stehen jedoch viele seltene CF-phänotypische Mutationen, für welche diese neuartigen Behandlungen nicht zugelassen sind, wodurch diese Mutationen von einer In-vitro-Analyse ihrer molekularen Konsequenzen profitieren würden. In-vitro-Untersuchungen von Membranproteinen werden oft durch die intrinsische Hydrophobizität und Aggregationsanfälligkeit dieser Proteine erschwert. Dies kann jedoch vermieden werden, indem kurze Membranproteinfragmente verwendet werden, die der kleinsten in vivo Faltungseinheit des jeweiligen Proteins an der ER-Membran entsprechen. Diese Modellproteine können routiniert genetisch verändert, exprimiert und aufgereinigt werden, was sie zu einem geeigneten Werkzeug macht, um die Auswirkungen von Mutationen zu genau festzustellen.
Diese Dissertation demonstriert die Nützlichkeit eines solchen reduktionistischen Modellsystems: TM3/4, das zweite helikale Haarnadel-Motiv der Transmembrandomäne 1 von CFTR, wurde verwendet, um Proteinfaltung mit Schwerpunkt auf krankheitsverursachende Missense-Mutationen von CFTR zu untersuchen, welche eine CFTR-Fehlfaltung in vivo verursachen können. Die TM3/4-Aufreinigung wurde zunächst durch die Verwendung eines Thioredoxin-Tags optimiert, der eine Hitzeaufreinigung des Fusionsproteins auch nach anfänglichen Reinigungsschritten ermöglichte. Die optimale Hitzebehandlung für maximale Proteinreinheit und -ausbeute wurde für TM3/4 und ein weiteres helikales Haarnadelprotein, die ATP-Synthase-Untereinheit c, bestimmt. Weiterhin wurde die tertiäre Faltung einer CF-phänotypischen Mutation, E217G, die ein nicht-natives GXXXG-Interaktionsmotiv einführt, mittels einzelmolekularem Förster-Resonanzenergietransfer (smFRET) in verschiedenen Lipiddoppelschichten analysiert, welche eine ungewöhnlich erhöhte Stabilität im Vergleich zum TM3/4-Wildtyp (WT) zeigte. Darüber hinaus wurde smFRET in Verbindung mit Circulardichroismus und Fluoreszenzspektroskopie verwendet, um die Wirkung eines spezifischen Membranlipids, Cholesterin, auf TM3/4-Varianten zu untersuchen, welches signifikante Auswirkungen auf die sekundäre, aber nicht auf die tertiäre Proteinstruktur hatte. Schließlich wurde eine Mutantenbibliothek von 13 TM3/4-Mutanten eingerichtet, um Wirkstoffscreenings mit CFTR-Korrektoren durchzuführen – einer Klasse kleiner Moleküle, die die Fehlfaltung von CFTR verhindern können. Diese Screening-Studie zeigte, dass (i) nicht alle CF-phänotypischen Missense-Mutationen lokal an einer Lipiddoppelschicht fehlgefaltet sind, die mit der ER-Membran vergleichbar ist; und (ii) die In-vitro-Wiederherstellung einer nativen WT-ähnlichen Konformation von lokal fehlgefalteten TM3/4-Mutanten ist nicht nur möglich, sondern es können auch verschiedene Wirkstoff-Mutanten-Paare identifiziert werden, die mit der Faltungsrettungseffizienz eines Korrektors auf eine bestimmte Mutante zusammenhängen. Die letztgenannten Wirkstoff-Mutanten-Paare können zu Drug-Repurposings führen, wenn die Wirkung in Zellkulturexperimenten bestätigt werden kann.
Im Allgemeinen, haben sich das TM3/4-Minimalfaltungsmodell von CFTR sowie biophysikalische Methoden, wie z.B. smFRET, als vielseitige Werkzeuge nicht nur für die Untersuchung von Mutations- und Lipideffekten auf die Membranproteinfaltung, sondern auch für das Screening von Medikamenten im Krankheitskontext erwiesen.:1 INTRODUCTION
2 THEORETICAL BACKGROUND
2.1 MEMBRANE PROTEINS AND THEIR NATIVE ENVIRONMENTS
2.1.1 Membrane protein families and their role in human health
2.1.2 Fundamental folding models of α-helical membrane proteins
2.1.3 Co-translational folding at the ER supported by the translocon
2.1.4 Folding-relevant interactions within membrane proteins
2.1.5 Biological membranes and lipid classes
2.1.6 Physical properties of lipid bilayers impacting membrane proteins
2.1.7 Membrane models for in vitro studies
2.2 CYSTIC FIBROSIS AND CFTR
2.2.1 Pathology of cystic fibrosis
2.2.2 Structure and function of the CFTR channel
2.2.3 A minimal model of CFTR to study rare CF mutations
2.2.4 Missense mutations within the CFTR segmental model TM3/4
2.2.5 Novel modulator therapies for the treatment of cystic fibrosis
2.3 IN VITRO ASSESSMENT OF MEMBRANE PROTEIN FOLDING
2.3.1 Expression and purification of membrane proteins
2.3.2 Single-molecule FRET in single- and multi-well mode for protein folding
3 HEAT PURIFICATION OF TRX MEMBRANE PROTEIN FUSIONS
3.1 PREAMBLE AND SUMMARY
3.2 RESULTS AND DISCUSSION
4 IMPACT OF A CFTR LOOP MUTATION WITH ATYPICAL STABILITY
4.1 PREAMBLE AND SUMMARY
4.2 RESULTS AND DISCUSSION
5 EFFECTS OF CHOLESTEROL ON LOCAL CFTR FOLDING
5.1 PREAMBLE AND SUMMARY
5.2 RESULTS
5.2.1 Folding of TM3/4 hairpins in the presence of cholesterol
5.2.2 Folding of TM3/4 hairpins in the presence of Lumacaftor
5.2.3 Impact of Lumacaftor on membrane fluidity
5.3 DISCUSSION
6 CFTR CORRECTOR SCREENINGS WITH SINGLE-MOLECULE FRET
6.1 PRESCREENING TO IDENTIFY MISFOLDED TM3/4 VARIANTS
6.2 SCREENING OF MISFOLDED TM3/4 VARIANTS WITH CFTR CORRECTORS
7 CONCLUSIONS
8 OUTLOOK
9 MATERIALS AND METHODS
9.1 CONSTRUCT DESIGN OF HELICAL TRANSMEMBRANE HAIRPINS
9.2 PROTEIN EXPRESSION AND PURIFICATION
9.3 HEAT TREATMENT OF HELICAL TRANSMEMBRANE CONSTRUCTS
9.4 SINGLE-MOLECULE FRET EXPERIMENTS
9.4.1 Labeling of TM3/4 constructs
9.4.2 Liposome preparation and reconstitution of labeled protein constructs
9.4.3 Single-molecule FRET measurements in manual mode
9.4.4 Single-molecule FRET measurements in multi-well screening mode
9.5 CIRCULAR DICHROISM SPECTROSCOPY
9.5.1 Circular dichroism to determine protein heat stability
9.5.2 Circular dichroism to study protein structure in different lipid bilayers
9.6 FLUORESCENCE SPECTROSCOPY
9.6.1 Vesicle leakage assay to test lipid bilayer stability
9.6.2 Examining lipid bilayer fluidity with fluorescent probes
10 APPENDIX
10.1 GENERATION OF A TM3/4 MUTANT LIBRARY
10.2 TM3/4 SCREENINGS WITH CFTR CORRECTORS
10.2.1 SmFRET control screenings and supporting data
10.2.2 Extracted closed state fractions from smFRET screenings
10.2.3 DLS to measure vesicle integrity after corrector addition
11 REFERENCES
12 ACKNOWLEDGEMENTS
13 ERKLÄRUNG GEMÄß §5 ABS. 1 S. 3 DER PROMOTIONSORDNUNG
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Advanced Fluorescence Microscopy to Study Plasma Membrane Protein DynamicsPiguet, Joachim January 2010 (has links)
Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane. / <p>QC 20151217</p>
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The Systematic Design and Application of Robust DNA BarcodesBuschmann, Tilo 19 September 2016 (has links) (PDF)
High-throughput sequencing technologies are improving in quality, capacity, and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag, index, or barcode that is attached to the sequencing or amplification primer and hence accompanies every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.
Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and codes based on the Levenshtein distance.
Levenshtein-based codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this thesis we demonstrate the decreased error correction capability of Levenshtein-based codes in a DNA context and suggest an adaptation of Levenshtein-based codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaptation, we take any DNA context into account and impose more strict rules for the selection of barcode sets. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors.
We present an adaptation of Levenshtein-based codes to DNA contexts capable of guaranteed correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of correcting on average more random mutations than traditional Levenshtein-based or Hamming codes. As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.
However, not every platform is susceptible to a large number of both indel and substitution errors. The Illumina “Sequencing by Synthesis” platform shows a very large number of substitution errors as well as a very specific shift of the read that results in inserted and deleted bases at the 5’-end and the 3’-end (which we call phaseshifts). We argue in this scenario that the application of Sequence-Levenshtein-based codes is not efficient because it aims for a category of errors that barely occurs on this platform, which reduces the code size needlessly. As a solution, we propose the “Phaseshift distance” that exclusively supports the correction of substitutions and phaseshifts. Additionally, we enable the correction of arbitrary combinations of substitution and phaseshift errors. Thus, we address the lopsided number of substitutions compared to phaseshifts on the Illumina platform.
To compare codes based on the Phaseshift distance to Hamming Codes as well as codes based on the Sequence-Levenshtein distance, we simulated an experimental scenario based on the error pattern we identified on the Illumina platform. Furthermore, we generated a large number of different sets of DNA barcodes using the Phaseshift distance and compared codes of different lengths and error correction capabilities. We found that codes based on the Phaseshift distance can correct a number of errors comparable to codes based on the Sequence-Levenshtein distance while offering the number of DNA barcodes comparable to Hamming codes. Thus, codes based on the Phaseshift distance show a higher efficiency in the targeted scenario. In some cases (e.g., with PacBio SMRT in Continuous Long Read mode), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.
For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.
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Confocal single-molecule fluorescence as a tool for investigating biomolecular dynamics in vitro and in vivoTorella, Joseph Peter January 2011 (has links)
Confocal single-molecule fluorescence is a powerful tool for monitoring conformational dynamics, and has provided new insight into the enzymatic activities of complex biological molecules such as DNA and RNA polymerases. Though useful, such studies are typically qualitative in nature, and performed almost exclusively in highly purified, in vitro settings. In this work, I focus on improving the methodology of confocal single-molecule fluorescence in two broad ways: (i) by enabling the quantitative identification of molecular dynamics in proteins and nucleic acids in vitro, and (ii) developing the tools needed to perform these analyses in vivo. Toward the first goal, and together with several colleagues, I have developed three novel methods for the quantitative identification of dynamics in biomolecules: (i) Burst Variance Analysis (BVA), which unambiguously identifies dynamics in single-molecule FRET experiments; (ii) Dynamic Probability Density Analysis (PDA), which hypothesis-tests specific kinetic models against smFRET data and extracts rate information; and (iii) a novel molecular counting method useful for studying single-molecule thermodynamics. We validated these methods against Monte Carlo simulations and experimental DNA controls, and demonstrated their practical application in vitro by analyzing the “fingers-closing” conformational change in E.coli DNA Polymerase I; these studies identified unexpected conformational flexibility which may be important to the fidelity of DNA synthesis. To enable similar studies in the context of a living cell, we generated a nuclease-resistant DNA analogue of the Green Fluorescent Protein, or “Green Fluorescent DNA,” and developed an electroporation method to efficiently transfer it into the cytoplasm of E.coli. We demonstrate in vivo confocal detection of smFRET from this construct, which is both bright and photostable in the cellular milieu. In combination with PDA, BVA and our novel molecular counting method, this Green Fluorescent DNA should enable the characterization of DNA and protein-DNA dynamics in living cells, at the single-molecule level. I conclude by discussing the ways in which these methods may be useful in investigating the dynamics of processes such as transcription, translation and recombination, both in vitro and in vivo.
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Structures and mechanical properties of single macromolecules at surfacesLiang, Hua 28 September 2015 (has links)
Drei ausgewählt makromolekulare Systeme: DNA, amphiphile Block-Bürstenpolymere, und amphiphile, hyperverzweigte Polyglycerine wurden untersucht, um die Zusammenhang zwischen Struktur, Eigenschaften, und potentiellen Anwendung auf der Ebene einzelner Moleküle zu widmen. Unterschiedliche DNA Konformationen: (i) supercoiled DNA, (ii) gestreckte doppelsträngige DNA, die teilweise in zwei Einzelstränge aufgeschmolzen ist, (iii) DNA mit einem überdehnten Rückgrat, (iv) entspannter, ungedehnter Ring und (v) kompaktes Knäuel wurden untersucht, um direkt DNA Konformationen mit mechanischen Eigenschaften, wie der Kopplung von Streckung und Verdrillung zu korrelieren. Mit Hilfe eines Kraftmikroskops, mit dem man eine Kraft parallel zur Oberfläche anlegen kann, wurden die plasmidischen DNA Moleküle auf bis zum 2.1-fachen der ursprünglichen B-Form Länge gestreckt und dann gerissen. Die Strukturen einzelner Amphiphilen Block-Bürstenpolymere auf unterschiedlichen Oberflächen wurden investigiert. Aus Chloroform-Lösung auf Glimmer abgeschiedene Polymere wiesen wurmartige Konformationen auf. Wegen der unterschiedlichen Oberflächenaffinitäten der Seitenketten sind diese zu einem Teil kollabiert, während sich ein anderer Teil ausstreckt. Das an Kaulquappen erinnernde Ergebnis ist eine Struktur mit rückgefalteten Kettenteilen. Aus wässriger Lösung abgeschieden bilden diese amphiphilen Block-Bürstenpolymere supramolekulare Aggregate auf der Oberfläche. Die amphiphile Kern-Schale-Strukturen der hyperverzweigten Polyglycerinen und ihre Verkapselungs- und Transportkapazität für typische Gastmoleküle wie Nil Rot und Pyren wurden mit Hilfe von SFM, Lichtstreu-, und Spektroskopie-Methoden examiniert. Die Ergebnisse zeigen, dass die Polymere verschiedene Gastmoleküle sowohl in unimolekulan Mizellen wie auch in polymeren Mizellen verkapseln und transportieren. Das Polymer ist ein vielversprechender Kandidat für die gleichzeitige Bereitstellung von zwei hydrophoben Pharmaka. / Three macromolecular systems: DNA, amphiphilic cylindrical polymer brushes, and amphiphilic core-shell structured hyperbranched polyglycerol (hPG) were investigated in order to investigate correlations between structure, properties and potential applications at the single molecule level. Different single DNA conformations: (i) supercoiled DNA, (ii) stretched DNA, partially melted into two single strands, (iii) DNA with an overstretched backbone, (iv) relaxed circles without stretching, and (v) compact coils were studied on the surface to directly correlate DNA conformations to mechanical properties such as twist-stretch coupling. The plasmid DNA molecules were stretched further, up to 2.1 times their original length and ruptured with a Scanning Force Microscope (SFM), exerting a force parallel to the surface. The structures of single cylindrical polymer brushes adsorbed on different surfaces were explored. The brush polymers reveal worm-like chain conformations on mica, after being deposited from a chloroform solution. Due to different affinities of the side chains to the surface, parts of the side chains collapsed, while others fully extended on the surface, resulting in a “tadpole like” or a back-folding structure. Deposited from an aqueous solution, the dual cylindrical polymer brushes form supramolecular aggregates on the surface. The supramolecular structure of hyperbranded polyglycerol and its encapsulation and transportation capacities for typical guest molecules, such as nile red and pyrene were examined by SFM, light scattering and spectroscopy methods. The polymer showed bi-functional carrier properties: it encapsulates and transports guest molecules in both, a “unimolecular micelle” and polymeric micelle type mechanism. The capacity of co-loading of two drugs and controlled release makes it a promising candidate for simultaneous delivery of two hydrophobic drugs in cancer combination therapy.
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The Systematic Design and Application of Robust DNA BarcodesBuschmann, Tilo 02 September 2016 (has links)
High-throughput sequencing technologies are improving in quality, capacity, and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag, index, or barcode that is attached to the sequencing or amplification primer and hence accompanies every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.
Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and codes based on the Levenshtein distance.
Levenshtein-based codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this thesis we demonstrate the decreased error correction capability of Levenshtein-based codes in a DNA context and suggest an adaptation of Levenshtein-based codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaptation, we take any DNA context into account and impose more strict rules for the selection of barcode sets. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors.
We present an adaptation of Levenshtein-based codes to DNA contexts capable of guaranteed correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of correcting on average more random mutations than traditional Levenshtein-based or Hamming codes. As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.
However, not every platform is susceptible to a large number of both indel and substitution errors. The Illumina “Sequencing by Synthesis” platform shows a very large number of substitution errors as well as a very specific shift of the read that results in inserted and deleted bases at the 5’-end and the 3’-end (which we call phaseshifts). We argue in this scenario that the application of Sequence-Levenshtein-based codes is not efficient because it aims for a category of errors that barely occurs on this platform, which reduces the code size needlessly. As a solution, we propose the “Phaseshift distance” that exclusively supports the correction of substitutions and phaseshifts. Additionally, we enable the correction of arbitrary combinations of substitution and phaseshift errors. Thus, we address the lopsided number of substitutions compared to phaseshifts on the Illumina platform.
To compare codes based on the Phaseshift distance to Hamming Codes as well as codes based on the Sequence-Levenshtein distance, we simulated an experimental scenario based on the error pattern we identified on the Illumina platform. Furthermore, we generated a large number of different sets of DNA barcodes using the Phaseshift distance and compared codes of different lengths and error correction capabilities. We found that codes based on the Phaseshift distance can correct a number of errors comparable to codes based on the Sequence-Levenshtein distance while offering the number of DNA barcodes comparable to Hamming codes. Thus, codes based on the Phaseshift distance show a higher efficiency in the targeted scenario. In some cases (e.g., with PacBio SMRT in Continuous Long Read mode), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives.
For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.
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Nanoscale Brownian Dynamics of Semiflexible BiopolymersMühle, Steffen 16 July 2020 (has links)
No description available.
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