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Comparative study of the performance of different spin labels for¹⁹F electron-nuclear double resonance (ENDOR) for short-range (1.0–1.5 nm) distance measurement in proteins. 

Abstract Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that Gd^III‐¹⁹F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell Gd^III‐¹⁹F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid Gd^IIItags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived Gd^III‐¹⁹F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the Gd^IIIand¹⁹F regions in the cell.