AFM characterization of mononucleosomes on surfaces

Introduction

The nucleosome core particle (NCP) is the foundation DNA-protein complex of chromatin, a structure into which the genomic DNA of all eukariotic species is packaged. The NCP consists of 147 base pairs of DNA wound in 1.7 left-handed superhelical turns around an octamer of four core histone proteins (H2A, H2B, H3 and H4). This unit is repeated along the DNA at an interval of about 170 to 220 base pairs [1]. Nucleosomes are structurally flexible, a property that is implicated in the regulation of all DNA-dependent processes [2].

Atomic force microscopy (AFM) can visualize biological samples under relatively mild sample preparation conditions and is suitable to reveal a structural flexibility of nucleosomes [3,4]. The figure below shows an example of an in vitro reconstituted nucleosome imaged by AFM in liquid [5]. However, a matter of discussion concerns the dependability of structures bound to surfaces, since the deposition process may distort nucleosomal conformations.

Project description

Here it is proposed to characterize systematically mononucleosomal conformations imaged by AFM. Specifically, three factors expected to influence imaged nucleosomal structures are of interest:

1) The ambient salt conditions, which modulate the nucleosome conformation due to electrostatic interactions.

2) The physico-chemical interaction with the surface used to immobilize the sample material.

3) The resolution of AFM-images, largely influenced by the used scanning probe.

You will learn to use AFM under different scanning conditions, i.e. in air and in liquid, and will also conduct biochemical sample preparations. In order to characterize sample-surface interactions, naked DNA conformations on surfaces will be analysed in parallel. To this end, basic concepts of polymer statistics will be applied. New results are expected that allow to better relate structures observed on surfaces to the suspected structures in free solution. The hope is to directly compare imaged nucleosomal structures with results of solution methods, like force-spectroscopy measurements under the same ambient conditions.

References

1. van Holde, K., (1989): Chromatin, Springer Verlag New York Inc.

2. Luger, K. & Hansen, J. (2005); Curr. Opin. Struct. Biol. 15 (2), 188-96.

3. Nikova, D.N., Pope, L.,Bennink, M., van Leijenhorst-Groener, K.A., van der Werf, K. and Geve, J. (2004); Biophys. J. 87, 4135-45.

4. Bussiek, M., Tóth, K, Brun, N, Langowski, J. (2005); J. Mol. Biol. 345 (4), 659-706.

5. Bussiek, M., Mücke, N., Müller, G., Diekmann, S., Waldeck, W., Langowski, J.; in preparation.