Extending the Range of Distances Accessible by 19F Electron-Nuclear Double Resonance in Proteins Using High-Spin Gd(III) Labels

Abstract

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.