Nanometer Scale Distance Measurements for Biological Systems using Gd$^{3+}$-based Spin Probes at High Magnetic Fields

Determination of nanometer-scale distances is critical for understanding structure and dynamics of proteins. Electron Paramagnetic Resonance (EPR), primarily below 1 T, is used to complement other structural techniques by quantifying sparse distances up to 8 nm in biomolecules labeled with nitroxide-based radicals. EPR becomes more powerful with increasing magnetic fields and frequencies. At 95 GHz (3.5 T), Gd$^{3+}$ ions have shown clear advantages over nitroxide probes (Potapov, JACS 2010). We show that these advantages are even more dramatic at 240 GHz (8.5 T). The width of Gd$^{3+}$'s central EPR transition narrows with increasing average distance between Gd$^{3+}$ ions out to distances as long as 5 nm. This doubles the distances accessible with nitroxides in continuous wave measurements, which can be carried out above the 200K protein-glass transition and with broad distance distributions. Temperature-dependent measurements of the phase memory times at 8.5 T and low temperatures show distance dependence out to 10 nm. Measurements of Gd$^{3+}$ labeled Proteorhodopsin confirm that phase memory times remain long enough to observe distance dependence in a spin-labeled protein. This work is supported by the National Science Foundation and the Binational Science Foundation.