Nanometer-range distance measurement in a protein using Mn ²⁺ tags

Pulse electron paramagnetic resonance measurements of long-range (nm scale) distances between spin labels site-specifically attached to biomacromolecules have proven highly effective in structural studies. The most commonly used spin labels are stable nitroxide radicals, and measurements are usually carried out at X-band frequencies (∼9.5 GHz, 0.35 T). Higher magnetic fields open new possibilities for distance measurements with increased sensitivity using alternative spin labels containing half-integer high-spin metal ions. Here we demonstrate W-band (95 GHz) pulse double electron-electron resonance (DEER) distance measurements in a protein labeled with two Mn ²⁺-EDTA tags. The distance distribution obtained is in excellent agreement with model calculations based on the known solution NMR structure. Thus, site-specific labeling with Mn ²⁺ tags opens a highly promising approach to nanometer distance measurements in biological macromolecules.