Prokopiou, GeorgiaLee, Michael D.Collauto, A.Abdelkader, ElwyBahrenberg, ThorstenFeintuch, AkivaRamirez Cohen, MarieClayton, JessicaSwarbrick, JamesGraham, BimOtting, GottfriedGoldfarb, Daniella2019-08-120020-1669http://hdl.handle.net/1885/164995The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd (S configuration) and C8-Gd (R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band 1H-ENDOR (electron–electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron–electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.D.G. acknowledges the support of the Israel Science Foundation (ISF grant No. 334/ 14). J.C. was supported by NSF grant MCB 1617025. This research was made possible in part by the historic generosity of the Harold Perlman Family (D.G.). D.G. holds the Erich Klieger Professorial Chair in Chemical Physics. T.B. acknowledges financial support from the Minerva Foundation. G.O. thanks the Australian Research Council (ARC) for a Laureate Fellowship, B.G. thanks the ARC for a Future Fellowship, and G.O. and B.G. thank the ARC for financial support.application/pdfen-AU© 2018 American Chemical Society201810.1021/acs.inorgchem.8b001332022-08-07