A New Gd³⁺ Spin Label for Gd³⁺-Gd³⁺ Distance Measurements in Proteins Produces Narrow Distance Distributions

Gd³⁺ tags have been shown to be useful for performing distance measurements in biomolecules via the double electron-electron resonance (DEER) technique at Q- and W-band frequencies. We introduce a new cyclen-based Gd³⁺ tag that exhibits a relatively narrow electron paramagnetic resonance (EPR) spectrum, affording high sensitivity, and which yields exceptionally narrow Gd³⁺-Gd³⁺ distance distributions in doubly tagged proteins owing to a very short tether. Both the maxima and widths of distance distributions measured for tagged mutants of the proteins ERp29 and T4 lysozyme, featuring Gd³⁺-Gd³⁺ distances of ca. 6 and 4 nm, respectively, were well reproduced by simulated distance distributions based on available crystal structures and sterically allowed rotamers of the tag. The precision of the position of the Gd³⁺ ion is comparable to that of the nitroxide radical in an MTSL-tagged protein and thus the new tag represents an attractive tool for performing accurate distance measurements and potentially probing protein conformational equilibria.