Selective Distance Measurements Using Triple Spin Labeling with Gd<sup>3+</sup>, Mn<sup>2+</sup>, and a Nitroxide

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron–electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the <i>Antennapedia</i> homeodomain orthogonally labeled with Gd<sup>3+</sup> and Mn<sup>2+</sup> tags in complex with its cognate DNA binding site labeled with a nitroxide.