ISBN 9783843906012

978-3-8439-0601-2, Reihe Physikalische Chemie

Wolfgang Kügel
Development and application of advanced single molecule fluorescence methods using PIE-MFD

255 Seiten, Dissertation Ludwig-Maximilians-Universität München (2012), Softcover, A5

Zusammenfassung / Abstract

This thesis focuses on the development of new advanced techniques for multiparameter fluorescence detection (MFD) experiments and shows first applications to important biological problems.

The main goal is to develop methods that yield reliable and precise single molecule FRET data to allow for a detailed insight into the shape and dynamics of biological complexes.

A new global approach to extract transition rates from fluorescence correlation spectroscopy (FCS) data is presented. Using this technique in combination with single-molecule burst analysis shows that the transition rates as well as the state distributions of double labeled DNA hairpins are influenced by the selected dye combination.

Moreover, single molecule burst analysis of MFD experiments is combined with the nano-positioning-system (NPS). NPS allows for the triangulation of unknown molecule parts in large biomolecules using a network of FRET based distance measurements. Here, the utilization of experimental FRET anisotropy data is for the first time demonstrated to clearly improve the positioning accuracy.

To this end new methods to allow for an unambiguous identification and separation of the desired population were developed. Furthermore, a robust way to extract residual anisotropy data as well as a new GPU based software implementation of the probability distribution analysis (PDA) is presented.

The last part consists of three sub projects that are in general all based on the application of single molecule burst analysis to recent questions regarding the various pathways that control the accessibility of nucleosomal DNA in a cell.

First, a method to obtain information about the remodeling throughput from single molecule data is introduced. This technique is then applied to questions regarding the two ISWI ATPases present in human cells, namely Snf2H and Snf2L.

The second nucleosome project is focused on the alternatively spliced histone variant H2A.Z.2.2. The experiments presented show that incorporation of H2A.Z.2.2 into nucleosomes leads to a significant destabilization compared to nucleosomes carrying H2A.Z.2.1 or canonical H2A.

Finally, nucleosomes can also be chemically modified to alter their stability. Here, nucleosomes either methylated or acetylated on the lysine at position 64 of the H3 histone are investigated and compared to unmodified nucleosomes for differences in their stability.