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. Such measurements allow access to distances too short to be measured 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 ¹⁹F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved ¹⁹F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. 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 may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.