Double-Arm Lanthanide Tags Deliver Narrow Gd -Gd Distance Distributions in Double Electron-Electron Resonance (DEER) Measurements.

Double-arm cyclen-based Gd tags are shown to produce accurate nanometer scale Gd -Gd distance measurements in double electron-electron resonance (DEER) experiments by confining the space accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α-helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd -Gd distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.