Regensburg 2013 – wissenschaftliches Programm

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O: Fachverband Oberflächenphysik

O 58: Poster Session III (Solid-liquid interfaces; Scanning probe and other methods; Electronic structure theory; Spin-orbit interaction)

O 58.49: Poster

Mittwoch, 13. März 2013, 18:15–21:45, Poster B1

Study of protein DNA interaction by simultaneous atomic force and fluorescence microscopy — •Sören Grannemann, Volker Walhorn, and Dario Anselmetti — Bielefeld University, Bielefeld, Germany

Atomic force microscopy (AFM) and fluorescence microscopy are in each case valuable tools for affinity studies at the single molecule level. A combination of both might even provide further insights in intermolecular binding and dissociation processes.

We combined AFM with total internal reflection microscopy (TIRFM) to study the affinity of a fluorescently labeled Protein DNA receptor ligand pair. The fluorescence markers were chosen such that Fluorescence Resonant Energy Transfer (FRET) can occur at close proximity of the dye molecules. Consequently, receptor-ligand binding should be correlated with FRET.

In first proof of principle experiments we used the DNA-binding Domain of the transcriptionfactor PhoB from E.coli and the associated DNA containing the PhoB recognition sequence as receptor-ligand system. Simultaneously acquired force and acceptor fluorescence data showed a correlation between the decrease of acceptor fluorescence intensity and molecular dissociation events of the Protein-DNA complexes. In present experiments, the donor fluorescence is measured additionally to prove the anti-correlation of donor and acceptor fluorescence intensity as well.