Spectroscopic selection of distance measurements in a protein dimer with mixed nitroxide and Gd ³⁺ spin labels

The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd ³⁺ tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd ³⁺ ion, and two Gd ³⁺ ions. Gd ³⁺-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.