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Multiparameter Fluoreszenzspektroskopie und mikroskopie zur Untersuchung biomolekularer Wechselwirkungen


Processes in cells can be attributed to the action of specific proteins on various levels. Thereby the concentration of an active protein at the right location is relevant, which opens up at least three principal pathways for the regulation of protein function on different levels: (I) regulation of gene expression (II) direct regulation of protein function via interconversion between different inactive and active protein conformations which can be spontaneous, induced by ligand binding or due to posttranslational modifications, (III) regulation of the local protein concentration via passive or active transport and compartmentalization of the cell.

To study these processes at high temporal and spatial resolution, especially fluorescence microscopy is a sensitive, specific and non-invasive tool that allows one to assess biochemical information. The combination of imaging methods with spectroscopic analysis allows one to collect many parameters simultaneously and thus increases the information content per image, which often is crucial for a correct conclusion of functional information.
The Seidel group will apply multi-parameter fluorescence detection (MFD) to study molecular aspects of transcriptional regulatory modules under in-vitro conditions and multi-protein complexes under invivo conditions.

In-vitro MFD studies.

Using a confocal fluorescence microscope the newly developed multiparameter fluorescence detection (MFD) enables us to simultaneously collect all fluorescence information such as intensity, lifetime, anisotropy in several spectral ranges) from picoseconds to seconds. MFD is applied to perform single-molecule fluorescence-resonance-energy-transfer (FRET) studies on nucleic acids labeled with a fluorescent donor and acceptor dye. Thus, it is possible to circumvent the classical pitfalls of the FRET method in ensemble measurements. These novel FRET-based detection and analysis methodologies allowed us to resolve structural subpopulations with sub-nanometer resolution.

Furthermore, direct access to the time trajectories of the different fluorescence parameters is obtained revealing the dynamics of the system. Finally, the construction of more-dimensional frequencyhistograms of the fluorescence parameters found in the trajectories on the single molecule level and selective analysis of these species (e.g. selective correlation analysis) give detailed view on the molecular energy landscape and the associated molecular structures.
Moreover a probability distribution analysis (PDA) method for calculating a priori histograms of FRET signals is presented taking explicitly crosstalk, stochastic variations and background into account. Histograms for the shot noise limited FRET signal are obtained by finding the mean as the only parameter in a least squares fit. Error analysis suggests an ultimate level of precision in 5 determining separations with FRET of 1% of the Förster radius. The PDA method unambiguously distinguishes between stochastic processes and broadening due to signal heterogeneity.

In this way quantitative structural information on various bent and kinked DNA and RNA structures was obtained. Moreover the structural and dynamic properties of the folding intermediate of a Holliday junction could be characterized. Finally single-molecule fluorescence studies on nucleic acid binding proteins will be discussed showing that MFD has developed to a powerful tool for Molecular Ångström Optics.

Multiparameter Fluorescence Imaging (MFI).

A new general strategy based on multiparameter fluorescence detection (MFD) is introduced to register and quantitatively analyse fluorescence images. Multiparameter Fluorescence Imaging (MFI) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. In addition the three spatial coordinates are stored. Statistically most efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels and not only for regions of interest. Their statistical significance is judged in a stack of two-dimensional histograms. In this way specific pixels can be selected for subsequent pixel based sub-ensemble analysis to improve statistical accuracy of the estimated parameters. MFI avoids sequential measurements, because the registered data allows one to perform many analysis techniques such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS) in an off-line mode. The limits of FCS to count molecules and to monitor dynamics are discussed. To demonstrate the ability of our technique, we analysed two model systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathionesepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3) (collaboration with U. Fleig (HHUD), which is involved in the dynamic cycle of polymerizing and depolymerising microtubules.


Publikationsliste ab 2004, Originalpublikationen (in press), bes. Publikationen aus Kooperationsprojekten

[s. Anl.]) sowie Patente

1. Diez, M., Zimmermann, B., Börsch, M., König, M., Schweinberger, E., Steigmiller, S., Reuter, R., Felekyan, S., Kudryavtsev, V. Seidel, C. A. M., Gräber, P.; Proton-powered subunit rotation in single membrane-bound F0F1- ATP synthase. Nat. Struct. Mol. Biol. 11, 135-141 (2004).

2. Schuette, C.G., Hatsuzawa, K., Margittai, M., Stein, A., Riedel, D., Küster, P., König, M., Seidel, C., Jahn, R.; Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc. Natl. Acad. Sci. USA. 101, 2858- 2863 (2004).

3. Oesterhelt, F., Schweinberger, E., Seidel, C.; Fluorescence labeling of RNA for single molecule studies, Handbook of RNA Biochemistry, Eds. Hartmann R. K., Bindereif A., Schön A., Westhof E., Wiley-VCH, Weinheim (2005) p. 453-474.

4. Eggeling, C., Volkmer, A., Seidel, C. A. M.; Molecular photobleaching kinetics of Rhodamine 6G under the conditions of one- and two-photon induced confocal fluorescence microscopy. ChemPhysChem 6, 791-804 (2005).

5. Gaiduk, A., Kühnemuth, R., Antonik, M., Seidel, C. A. M.; Optical characteristics of AFM tips for single molecule fluorescence applications. ChemPhysChem 6, 976-983 (2005).

6. Al-Soufi, W., Reija, B., Novo M., Felekyan, S., Kühnemuth, R., Seidel, C. A. M., Fluorescence correlation spectroscopy, a tool to investigate supramolecular dynamics: Inclusion complexes of pyronines with cyclodextrines. J. Am. Chem. Soc. 127, 8775-8784 (2005).

7. Felekyan, S., Kühnemuth, R., Kudryavtsev, V., Sandhagen, C., Becker, W., Seidel, C. A. M.; Full correlation from picoseconds to seconds by time-correlated single photon detection. Rev. Sci. Instr. 76, 083104 (2005).

8. Wo?niak, A. K., Nottrott, S., Kühn-Hölsken, E., Schröder, G. F., Grubmüller, H., Lührmann, R., Seidel, C. A. M., Oesterhelt F.; Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements. RNA, 11, 1545-1554 (2005).

9. Widengren, J., Kudryavtsev, V., Antonik, M., Berger, S., Gerken, M., Seidel, C. A. M.; Single molecule detection and identification of multiple species by multiparameter fluorescence detection. Anal. Chem. 78, 2039-2050 (2006).

10. Eggeling, C., Widengren, J., Brand, L., Schaffer, J., Felekyan, S., Seidel, C. A. M.; Analysis of photobleaching in single-molecule multicolour excitation and fluorescence resonance energy transfer measurements. J. Phys. Chem. A 110, 2979-2995 (2006).

11. Antonik, M., Felekyan, S., Gaiduk, A., Seidel, C. A.M.; Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis. J. Phys. Chem. B 110, 6970-6978 (2006).

12. Gaiduk, A., Kühnemuth, R., Felekyan, S., Antonik, M., Becker, W., Kudryavtsev, V., König, M., Oesterhelt, F., Sandhagen, C. Seidel C.A.M.; Time-resolved photon counting allows for new temporal and spatial insights into the nanoworld. Proc. of SPIE- Int. Soc. Opt. Eng 6372 (Advanced Photon Counting Techniques, edited by

Wolfgang Becker), 637203 (2006).·

13. Kudryavtsev, V., Felekyan, S., Wo?niak, A. K., König, M., Sandhagen, C., Kühnemuth, R., Seidel C. A.M., Oesterhelt, F.; Multiparameter fluorescence imaging to monitor dynamic systems, Anal. Bioanal. Chem. 387, 71- 82 (2007) DOI 10.1007/s00216-006-0917-0.

14. Widengren, J., Chmyrov, A., Eggeling, C., Löfdahl, P.-Å., Seidel, C. A. M.; Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J. Phys. Chem. A 111, 429-440 (2007).

15. Novo, M., Felekyan S., Seidel C.A.M. , and Al-Soufi, W.; Dye-Exchange Dynamics in Micellar Solutions studied by Fluorescence Correlation Spectroscopy. J. Phys. Chem. B 111, 3614-3624 (2007).

16. Gaiduk, A., Kühnemuth, R., Felekyan, S., Antonik, M., Becker, W., Kudryavtsev, V., Sandhagen, C. Seidel C.A.M.; Fluorescence detection with high time-resolution: From optical microscopy to simultaneous force and fluorescence spectroscopy. Microscopy Research and Technique, 70, 433-441 (2007).

17. Neubauer, H., Gaiko, N., Berger, S., Schaffer, J., Eggeling, C., Verdier, L. Seidel, C. A. M., Griesinger C., Volkmer, A.; Orientational and dynamical heterogeneity of Rhodamine 6G terminally attached to a DNA helix revealed by NMR and single-molecule fluorescence spectroscopy. J. Am. Chem. Soc. 129, 12746-12755 (2007).

18. Kalinin, S., Felekyan, S. Antonik, M., Seidel C. A. M. Probability Distribution Analysis of Single-Molecule Fluorescence Anisotropy and Resonance Energy Transfer. J. Phys. Chem. B 111, 10253-10262 (2007).