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 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 Gd III and 19F regions in the cell.
| Original language | English |
|---|---|
| Article number | e202218780 |
| Journal | Angewandte Chemie (International ed.) |
| Volume | 62 |
| Issue number | 20 |
| DOIs | |
| Publication status | Published - 8 May 2023 |
Funding
We thank Mr. Ciarán Rogers, University of Manchester, for his participation in some of the initial solution ENDOR measurements, and W.Z. and A.M.G. thank Doug Bevan for computer and Michael J. Delk for NMR technical support. This work was funded by the National Science Foundation USA-Israel Science Foundation program through BSF 2021617 (to D.G.), the NSFC-ISF China-Israel 3559/21 Research Grant Program (to D.G.) and NSF-MCB 2116534 to (A.M.G.), as well as NSF grant CHE 1708773 (to A.M.G.) and made possible, in part, by the historic generosity of the Harold Perlman Family (D.G.). D.G. holds the Erich Klieger Professorial Chair in Chemical Physics.