The design, synthesis, and application of a nine-coordinate gadolinium(III)-containing spin label, [Gd.sTPATCN]-SL, for use in nanometer-distance measurement experiments by EPR spectroscopy is presented. The spin label links to cysteines via a short thioether tether and has a narrow central transition indicative of small zero-field splitting (ZFS). A protein homodimer, TRIM25cc, was selectively labeled with [Gd.sTPATCN]-SL (70%) and a nitroxide (30%) under mild conditions and measured using the double electron electron resonance (DEER) technique with both commercial Q-band and home-built W-band spectrometers. The label shows great promise for increasing the sensitivity of DEER measurements through both its favorable relaxation parameters and the large DEER modulation depth at both Q- and W-band for the inter-Gd(III) DEER measurement which, at 9%, is the largest recorded under these conditions.
Bibliographical noteFunding Information:
The authors thank the ERC (266804), EPSRC (EP/R013705/ 1, EP/M508214/1), and the Carnegie Trust (RIG007510) for financial support, the Wellcome Trust for a Multi-User Equipment Grant (099149/Z/12/Z), and the Royal Society for a University Research Fellowship to JEL. We also thank Professor Catherine Botting and Dr. Sally Shirran for assistance with MALDI TOF at St Andrews.
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