|Katrin Schulze, Silke Hutschereiter, Suman Lata, Jacob Piehler, and Robert Tampé
Institute of Biochemistry, Biocenter, Marie-Curie-St. 9, D-60439 Frankfurt/Main, Germany
The manipulation and labelling of biomolecules with biochemical tweezers is an expedient and versatile tool for the functional analysis of biomolecules. Here we used tweezers consisting of several Ni-NTA-complexes which can interact with different His-tags. The multivalency of these tweezers leads to a specific, reversible and stable binding between the chelator and the His-tags.
When tagged with a fluorescent dye, these tweezers can be used for labelling of functional proteins to investigate molecular processes. Size exclusion chromatography experiments showed a stable binding between these probes and the functional biomolecules. These fluorescent probes facilitate the site-specific, stoichiometric and reversible labelling of proteins in vitro and in vivo. The fluorescence signal enables a simple and efficient read-out by fluorescence spectroscopy and microscopy.
Untagged multivalent NTA-chelators have been used successfully to manipulate the 20 S proteasome. The proteasome, which plays an important role in the cell, eliminates misfolded and malfunctioning proteins by degradation. With the multivalent chelators it was possible to manipulate the α-N His-tagged proteasome, whereas the β-C His-tagged proteasome was not affected. Hence, some of these chelators can be used to switch the activity of the α-N His-tagged proteasome.