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The Global ETD Search service is a free service for researchers
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Networked Digital Library of Theses and Dissertations.
Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
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Mechanical unfolding of membrane proteins captured with single-molecule AFM techniquesBaltrukovich, Natalya 08 January 2009 (has links) (PDF)
Atomic force microscopy (AFM) is a powerful technique that enables to study biological macromolecules and dynamic biological processes at different scales. It is an excellent tool for imaging of biological objects under various conditions at a nanometer resolution. Force mode of AFM, so called single molecule force spectroscopy (SMFS), allows for investigation of the strength of molecular interactions of different origins established between and within biological molecules. In the present work, SMFS was used to detect and locate structurally and functionally important interactions of sodium/glycine betaine transporter BetP of Corynebacterium glutamicum, which serves as a model system for this class of proteins. Mechanical pulling of BetP molecules embedded into the lipid membranes resulted in a step-wise unfolding of the protein and revealed insights into its structural stability. Effect of the lipid environment, N- and C-terminal extensions on inramolecular interactions of BetP as well as protein activation and ligand binding were investigated in great detail. In another part of this work, I demonstrate an application of the AFM based technique that can record unfolding of a protein under force-clamp conditions. This method directly measures the kinetics of the protein unfolding, allowing for the use of simple methods to analyze the data. For the first time the force-clamp technique was used to describe in detail unfolding kinetics of the membrane protein, i. e. Na+/H+-antiporter NhaA from Escherichia coli. Performed here experiments on NhaA in its functionally active and inactive states demonstrated the advantages of examining unfolding kinetics at the single-molecule level. It was possible to observe unfolding events for pH-activated conformation of NhaA that due to the low frequency of occurrence were not represented in the ensemble average of the single-molecule measurements. As mechanical unfolding, similarly to bond rupture, is a force-dependent process, force-clamp technique can allow for a more direct way of probing protein unfolding and is anticipated to be also useful to examine the folding/unfolding kinetics of other membrane proteins.
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Investigation of biological macromolecules using atomic force microscope-based techniquesBippes, Christian Alexander 19 August 2009 (has links) (PDF)
The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques.
In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies.
A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods.
A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed.
Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane.
Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.
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Entwicklung eines miniaturisierten Ionenfilters und Detektors für die potentielle Anwendung in IonenmobilitätsspektrometernGraf, Alexander 22 May 2015 (has links) (PDF)
Die Ionenmobilitätsspektrometrie ermöglicht eine selektive Detektion von niedrigkonzentrierten Gasen in Luft. Darauf beruhende Analysegeräte können verhältnismäßig einfach umgesetzt werden und in vielfältigen mobilen Einsatzszenarien wie der Umweltanalytik Anwendung finden.
Die vorliegende Dissertation gibt einen Überblick über die Grundlagen der Ionenmobilitätsspektrometrie und setzt die funktionellen Teilkomponenten Ionenfilter und Ionendetektor mit Mikrosystemtechniken um. Dafür werden Möglichkeiten aus dem Stand der Technik vorgestellt und eine für die Umsetzung optimale Variante identifiziert. Ein Ionenfilter basierend auf der Differenzionenmobilitätsspektrometrie zeigt diesbezüglich ein sehr geeignetes Skalierungsverhalten.
Zur Integration in einen Demonstrator-Chip wird ein neuartiges Bauelementkonzept verfolgt, mit technologischen Vorversuchen untersetzt und erfolgreich in einen Gesamtherstellungsablauf überführt.
Mit Hilfe von weiterführenden analytischen Untersuchungen werden spezifische Phänomene bei der elektrischen Kontaktierung der verwendeten BSOI-Wafer als Ausgangsmaterial hergeleitet und Empfehlungen zur Vermeidung gegeben.
Der Funktionsnachweis der Teilkomponente Ionendetektor wird anhand von hergestellten Demonstrator-Chips und mit Hilfe eines entwickelten Versuchsaufbaus begonnen.
Es werden die weiteren Schritte zum Nachweis der Gesamtfunktionalität abgeleitet und festgehalten.
Auf Basis des umgesetzten Bauelement- und Technologiekonzepts und der vorliegenden Ergebnisse, wird das entwickelte und realisierte Gesamtkonzept als sehr aussichtsreich hinsichtlich der favorisierten Verwendung als Teilkomponente eines miniaturisierten Ionenmobilitätsspektrometers eingeschätzt.
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