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.

Effective assessment of lanthanide ion delivery into live cells by paramagnetic NMR spectroscopy

“…Only the signals from endogenous Mn 2+ were determined, consistent with previous in-cell reports. 4 Notably, the EPR spectra for the cells with expressed LanM-GB1 displayed distinct signals of LanM-GB1-Gd (Fig. 4).…”

“…1 Lanthanide ions (Ln 3+ ) are the focus in the fields of biomedical analysis, imaging diagnosis, and structural characterization of proteins in cells. 2–4 Recently, Ln 3+ have been reported to confer some biological functions in several methylotrophs. 5…”

“…8 The concentrations of protein–Ln 3+ complexes in vivo are estimated using the NMR or EPR spectra, generally in about tens of μM. 3,4 However, the invasive delivery of protein–Ln 3+ complex into cells is not suitable for most of the endogenetic targets.…”

Structure and Flexibility of Copper‐Modified DNA G‐Quadruplexes Investigated by 19F ENDOR Experiments at 34 GHz**

“…Electron-nuclear double resonance (ENDOR) spectroscopy, combined with 19 F labeling, has been used to determine electron-nuclear distances for atomic-scale structure elucidation of various biopolymers. [29][30][31][32] This technique has been applied to 19 F-equipped biological macromolecules ranging from spinlabeled RNA to radical-containing protein machineries [33][34][35][36][37] in the last three years. However, all of these recent studies were carried out at the microwave (mw) frequency of 94 GHz (Wband) or higher, where the overlap with proton ENDOR resonances is avoided or minimized.…”

19F electron nuclear double resonance (ENDOR) spectroscopy for distance measurements using trityl spin labels in DNA duplexes

“…On the other hand, orientation selection can lead improved sensitivity for very small couplings, by separating different components of the dipolar tensors, and provides more information about the system. One way to avoid orientation selection is to use Gd 3+ complexes tags, as proposed by D. Goldfarb and coauthors, [19][20][21] which work best in high magnetic fields starting from W-band The main advantage of Gd 3+ is its narrow central EPR transition at high magnetic fields. These labels have also provided high-sensitivity ENDOR signals at physiological concentrations (B5 mM).…”