|Annett Reichel, Jennifer Strunk, Suman Lata, Dirk Schaible, and Jacob Piehler
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt a.M., Germany
Probing protein function such as interactions with other proteins or conformational changes requires tools for site-specific modification. We have engineered supramolecular entities containing 2-4 nitrilotriacetic acid (NTA) moieties, which bind oligohistidine tags with high affinity and stability. Stable and stoichiometric labeling of histidine-tagged proteins was achieved by tris- and tetrakis-NTA, enabling for versatile application in functional proteins analysis. Here, we demonstrate functional immobilization of proteins on membranes for exploring lateral protein interactions in cytokine receptor assembly. We present versatile assays for probing the binding affinities and the interaction kinetics by making use of site-specific tethering proteins onto the membrane. Furthermore, tris-NTA was employed for non-covalent labeling of proteins with different fluorescence dyes. Using these tools for site-specific dual color labeling we explored the conformation dynamics and ligand-induced conformational changes of proteins by Förster resonance energy transfer (FRET). Tris-NTA/fluorophore conjugates were employed for site-specifically incorporating a FRET acceptor into the protein in situ. Thus, the efficiencies of intramolecular FRET were quantified reliably, and ligand-induced conformational changes were probed by changes in fluorescence intensity and life-time. Furthermore, the conformational dynamics of the protein was followed by fluorescence correlation spectroscopy.