Gd(III)‐19F Distance Measurements of Proteins in Cells by Electron‐Nuclear Double Resonance

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