Gd^III‐¹⁹F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

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 GdIII‐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 GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐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 GdIII and 19F regions in the cell.

Extending the range of distances accessible by 19F electron-nuclear double resonance in proteins using high-spin Gd(III) labels

“…Going beyond in vitro measurements, in-cell Gd(III)- 19 F ENDOR significantly expanded the scope of this technique. 27 When applied to the same systems, 19 F ENDOR data is complimentary to results obtained by 19 F paramagnetic relaxation enhancement (PRE) and pseudo contact shift (PCS) solution nuclear magnetic resonance (NMR) techniques . [34][35][36] The latter cover similar distance ranges, are carried out near room temperature and provide average electronnuclear distances, while ENDOR measurements are performed in the frozen state and report on the conformational distribution.…”

“…Proteins were prepared and spin-labeled as described previously. 27 Ubiquitin T66C possesses 4-trifluoromethyl phenylalanine (tFmPhe) at position 45 and GB1 Q32C contains 5-fluorotryptophan (5F-Trp) at position 43. The BrPSPyDO3A-Gd(III) tag was attached to the single cysteines on both proteins.…”

“…Given the excellent results for the synthetic ruler, we proceeded to apply the above detailed methodology to two protein samples with low resolution 19 F ENDOR spectra at the CT arising from longer Gd-F distances and possibly broader Gd-F distance distributions. 27…”

“…Traditionally, it has been applied to extract the local spatial and electronic structure of intrinsic paramagnetic metal ions or metal clusters in proteins, [15][16][17][18][19][20] while, at present, ENDOR experiments for distance determination on spin labeled biomolecules are being developed. [21][22][23][24][25][26][27] For measuring weak hyperfine interactions, which are dipolar in nature, Mims ENDOR 28 is the technique of choice, as demonstrated for a nitroxide label situated 1 nm away from a 31 P nucleus in a membrane bilayer. 29 Bennati and co-workers demonstrated on synthetic models and RNA molecules that distances up to 1.5 nm (15 Å) can be determined by combining nitroxide and 19 F labeling.…”

Gadolinium Spin Decoherence Mechanisms at High Magnetic Fields

“…At high magnetic fields B 0 , this central transition narrows as 1/ B 0 , leading to field-dependent improvements in sensitivity and resolution. 12 , 17 , 18 Long-range interactions between Gd 3+ centers are stronger than between spin-1/2 centers, because Gd 3+ possesses a large magnetic moment of 7 times that of a spin-1/2 system. At high magnetic fields and cryogenic temperatures, the spin–lattice relaxation characterized by the time constant T 1 and the spin decoherence time T M are both longer than those for most high-spin metal ions.…”