Interspin distances between 0.8 nm and 2.0 nm can be measured through the dipolar broadening of the continuous wave (cw) EPR spectrum of nitroxide spin labels at X-band (9.4 GHz, 0.35 T). We introduce Gd3+ as a promising alternative spin label for distance measurements by cw EPR above 7 Tesla, where the |−1/2〉 to |1/2〉 transition narrows below 1 mT and becomes extremely sensitive to dipolar broadening. To estimate the distance limits of cw EPR with Gd3+, we have measured spectra of frozen solutions of GdCl3 at 8.6 T (240 GHz) and 10 K at concentrations ranging from 50 mM to 0.1 mM, covering a range of average interspin distances. These experiments show substantial dipolar broadening at distances where line broadening cannot be observed with nitroxides at X-band. This data, and its agreement with calculated dipolar-broadened lineshapes, show Gd3+ to be sensitive to distances as long as ∼3.8 nm. Further, the linewidth of a bis-Gd3+ complex with a flexible ∼1.6 nm bridge is strongly broadened as compared to the mono-Gd3+ complex, demonstrating the potential for application to pairwise distances. Gd-DOTA-based chelates that can be functionalized to protein surfaces display linewidths narrower than aqueous GdCl3, implying they should be even more sensitive to dipolar broadening. Therefore, we suggest that the combination of tailored Gd3+ labels and high magnetic fields can extend the longest interspin distances measurable by cw EPR from 2.0 nm to 3.8 nm. cw EPR data at 260 K demonstrate that the line broadening remains clear out to similar average interspin distances, offering Gd3+ probes as promising distance rulers at temperatures higher than possible with conventional pulsed EPR distance measurements.
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