Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR.
Benesh JosephVictor M TormyshevOlga Yu RogozhnikovaDmitry AkhmetzyanovElena G BagryanskayaThomas F PrisnerPublished in: Angewandte Chemie (International ed. in English) (2016)
The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.