Welegedara, Adarshi PYang, YinLee, Michael DSwarbrick, James DGraham, BimGoldfarb, DaniellaOtting, GottfriedHuber, Thomas2018-10-182018-10-180947-6539http://hdl.handle.net/1885/148487Double-arm cyclen-based Gd3+ tags are shown to produce accurate nanometer scale Gd3+ -Gd3+ 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 Gd3+ -Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double-arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.Financial support by the Australian Research Council is gratefully acknowledged. D. G. acknowledges the support of the Israel Science Foundation (ISF grant No. 334/14). 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.application/pdfhttp://www.sherpa.ac.uk/romeo/issn/0947-6539/..."author can archive post-print (ie final draft post-refereeing). 12 months embargo" from SHERPA/RoMEO site (as at 18/10/18). This is the pre-peer reviewed version of the following article: [Welegedara, Adarshi P., et al. "Double‐Arm Lanthanide Tags Deliver Narrow Gd3+–Gd3+ Distance Distributions in Double Electron–Electron Resonance (DEER) Measurements." Chemistry–A European Journal 23.48 (2017): 11694-11702.], which has been published in final form at [https://dx.doi.org/10.1002/chem.201702521]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versionsepr spectroscopydouble electron-electron resonancegadoliniumprotein structures2017-08-2510.1002/chem.201702521