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 ( 19 F 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 19 F ENDOR almost 2fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved 19 F 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 19 F and Gd(III), in which the Gd−F distance is too long to produce a well-resolved 19 F 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 19 F ENDOR approach may be applicable to Gd−F distances as large as 20 Å, widening the current ENDOR distance window.

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy

“…In solution, measurements of distances up to 24 Å to a paramagnetic nitroxide label have been reported . Recently developed EPR methods have measured accurate distances in the range of 5–20 Å with exquisite sensitivity that enables in-cell analyses of proteins at low concentrations. − In protein engineering, F-Trp residues have been used to modify protein properties . An exciting recent finding is that 7F-Trp can form photoinduced fluorescent protein cross-links with phenylalanine and tyrosine …”

Fluorinated Tags to Study Protein Conformation and Interactions Using 19F NMR

“…19 F signal perturbations gave an insight into the association of the two proteins and the structural changes that govern this. Recently, a related approach incorporated a trifluoromethylphenylalanine and a high-spin Gd(III) paramagnetic tag as a 'molecular ruler' for long range 19 F electron-nuclear double resonance (ENDOR) distance measurements between the spin tag and a fluorinated reporter, [65] which is effective up to a distance of 25 Å, [66] as an alternative to more traditional EPR-based distance measurements in biomolecules.…”