Browsing by Subject "TIRF-Mikroskopie"

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Untersuchung einer Methode zur spezifischen Fluoreszenz – Markierung von Signalproteinen und deren Beobachtung in lebenden Escherichia coli (Einzelmolekültechnik & Perspektiven)
(2016) Ehrhard, Tanja Margret; Herten, Dirk-Peter
Malfunctions in signal transduction often cause diseases such as cancer and metabolic disorders. A thorough understanding of the relevant mechanisms of signal transduction is therefore an important requirement for the development of therapies and pharmaceuticals. In this thesis, a method was developed, which allows the observation of individual signaling proteins and their interactions in living cells. Therefore this method has advantages compared to molecular detection methods which are based on ensemble averages. As a model system for signal transduction, the bacterial chemotaxis with its regulator protein CheY was selected. The experimental studies were carried out with total internal reflection fluorescence microscopy (TIRFM), which requires a fluorescent labeling of the examined molecules. To ensure a specific and background reduced labeling, bright and photostable fluorescent ,tags are needed. In this work, the SNAP-tag system was used, which allows the use of different dyes. An advantage of this system is the possibility of using fluorescence-quenched benzyl guanine (BG)-dyes, which show a strong fluorescence only after binding to SNAP-tag. For development of the labeling method, the dyes Atto 620, Atto 633, Atto 655 and Atto 680 were analyzed in preliminary experiments regarding their fluorescence, photostability and blinking behavior. The thorough knowledge of these properties is essential for the correct interpretation of the experimental results. Dyes which are ideal for the method have a high fluorescent signal over a long observation time, and they are stable and do not interfere with the function of the target molecule. The preliminary investigations have shown that among the dyes tested, Atto 633 had the best photophysical properties for labeling with the SNAP-tag system and also the best cell permeability. This allows, under continuous laser excitation, to observe individual molecules for several seconds. In addition, the labeling efficiency was controlled by the protein expression, the dye concentration, and the incubation time of the dye. For single-molecule detection, a low labeling efficiency is of advantage since too high density of fluorescently labeled molecules makes the identification of individual molecules difficult. Subsequently, a labeling protocol was established which allows a specific, background- reduced fluorescence labeling of individual CheY proteins in living E. coli cells, without impairment of the protein’s functionality. Real-time detection with a time resolution of 30 milliseconds showed that it is possible to observe individual CheY molecules as a fluorescent point during the state of binding to an interaction partner. By means of numerical methods, the state of binding can be extracted from the fluorescence intensity traces as on/ off and their probability distribution can be determined. These quantitative studies gave indications on specific protein interactions, but no detailed information on binding times could be found. Different interactions of the protein, both specific and non-specific nature, could be the reason. Therefore, another important development of this labeling system would be the opportunity of simultaneous staining of two or more proteins with spectroscopically distinguishable fluorescent tags (e.g. CLIP-tag) to perform colocalization with alternating laser excitation. Another cause might be found in the nature of the dye itself. Laser- and temperature-dependent studies could provide further information concerning the behavior of the dyes. Thus, the described fluorescence labeling method provides a new approach for quantitative studies of protein interactions in living cells.