|
Upcoming events |
|
Program
1. Welcome 2. Alex Gansen (DKFZ) - "Chromatin at the nanolevel: single molecule spectroscopy of individual nucleosomes" The local structure and dynamics of nucleosomes affects the subsequent compaction into the higher order chromatin structure, which is an important regulation mechanism of gene activity in eukaryotic cells. To follow dynamic changes in the structure of nucleosomes we apply the method of Fluorescence Resonance Energy Transfer (FRET) in ensemble and on the single molecule level. The power of single molecule methodologies lies in the ability to resolve subpopulation within a mixture. We utilize a custom build confocal microscope system to investigate the dynamics within individual nucleosomes diffusing free in solution. DNA labelled with Alexa488 and various acceptor dyes (Rhodamine X, Alexa568, Alexa594) was reconstituted onto histone octamers to form nucleosomes with 20 80 % average energy transfer.. Fluorophore position was chosen to monitor different parts of the nucleosomes. This talk will discuss the principles of the method and the aspects which have to be considered when studying individual nucleosomes under single molecule conditions. Several examples will be presented which indicate the power and the limitations of the existing approach. These will include results on the local dynamics of internal DNA sites and some preliminary data on the thermally and enzymatically induced repositioning. In eukaryotic cells, DNA is packed with histone proteins into chromatin. Because many biological processes require a free DNA as a substrate, the chromatin structure needs to transit between tight and loose states. To investigate this long time-scale dynamical process, we performed coarse-grained molecular dynamics on the nucleosome, the basic unit of chromatin. Here we present a method for parameterizing the force field for this model based on a Boltzmann inversion of the radial distribution function obtained from crystallographic and all-atom molecular dynamics data. We derived a set of parameters which enabled us to perform coarse-grained molecular dynamics whose fluctuations are in accord with the ones derived from all-atom MD. |