Extending the range of distances accessible by 19F electron-nuclear double resonance in proteins using high-spin Gd(III) labels

Fluorine electron-nuclear double resonance (19F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either intrinsic or conjugated to a biomolecule via spin labeling, yielding distances beyond those accessible by double electron-electron resonance (DEER). To further extend the accessible distance range we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|–1/2> <-> |+1/2>), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of ENDOR almost twofold. We first demonstrate quantitative agreement between the experimental spectra and theoretical predictions for a model fluorine containing Gd(III) complex, whose 19F spectrum is well resolved in conventional central transition measurements. We then validate our approach on two proteins labeled with 19F and Gd(III), in which the Gd-F distance is too long to produce a well resolved 19F ENDOR doublet when measured at the central transition. By focusing on the |–5/2> <-> |–3/2> and |–7/2> <-> |–5/2> EPR transitions, a resolution enhancement of 4.5 and 7 fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended 19F ENDOR approach should be applicable to Gd-F distances as large as 20Å, widening the traditional ENDOR distance window.