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 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(III) tags. 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 cell.