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978-3-86853-864-9, Reihe Physikalische Chemie
Advanced Fluorescence Fluctuation Spectroscopy with Pulsed Interleaved Excitation
141 Seiten, Dissertation Ludwig-Maximilians-Universität München (2011), Softcover, A5
In this work, a wide spectrum of Fluorescence Fluctuation Spectroscopy (FFS) methods was implemented, optimized, and applied to relevant biophysical systems. Scanning FCS and RICS, two FFS techniques specially suited for live-cell measurements, were combined with Pulsed Interleaved Excitation. The selective crosstalk-free cross-correlation removes the inherent flaw of these methods that showed a non-zero cross-correlation even for non-interacting species. The necessity for an argumentation based on different cross-correlation amplitudes to show molecular binding is omitted, and an immediate, unambiguous statement becomes possible without the need for a binding-free reference measurement. Successful proof-of-principle experiments were performed both in aqueous solutions and in living cells. Fluorophores with correlated and with uncorrelated motion served as positive and negative controls, respectively. The new capabilities of PIE-RICS were then applied by investigating the binding characteristics of Calmodulin to the CaV1.4 calcium channel. The cytosolic part of the channel, labeled with mCherry, was expressed in HEK293 cells together with GFP-labeled Calmodulin. Under the chosen experimental conditions, no binding could be observed. Without PIE, such a clear statement would have been impossible. In a second assay, the inhibitory function of the ICDI domain of the CaV1.4 channel was studied. The GFP-labeled C-terminal part of the CaV1.4 channel without its ICDI domain and the isolated ICDI-domain itself, labeled with mCherry, were expressed in HEK293 cells. Again, no binding could be observed between those two species under the present conditions.
In addition to these correlation-based methods, there are a variety of fluorescence brightness analysis methods: The Photon Counting Histogram (PCH), Fluorescence Intensity Distribution Analysis (FIDA), and Fluorescence Cumulant Analysis (FCA). These methods were carefully evaluated with regard to their reliability and accuracy. FIDA proved to be the best method for brightness analysis, with FCA also delivering accurate results for experiments with only one fluorescent species. Brightness analysis was then applied to the interaction of DNA methyl transferase 1 (Dnmt1) with substrate DNA strands. Dnmt1 was previously suspected to form dimers in the active state. With all brightness analysis methods, however, no dimers were found. Furthermore, brightness analysis indicated multiple DNA substrates per Dnmt1 molecule. This surprising result was further investigated with Fluorescence Cross-Correlation Spectroscopy (FCCS) in combination with PIE. Both the monomeric state of Dnmt1 and the complexation with multiple DNA strands could be confirmed with PIE-FCCS.