Gd(III)–Gd(III) Relaxation-Induced Dipolar Modulation Enhancement for In-Cell Electron Paramagnetic Resonance Distance Determination

Abstract

In-cell distance determination by electron paramagnetic resonance (EPR) spectroscopy reveals essential structural information about biomacromolecules under native conditions. We demonstrate that the pulsed EPR technique RIDME (relaxation induced dipolar modulation enhancement) can be utilized for such distance determination. The performance of in-cell RIDME has been assessed at Q-band using stiff molecular rulers labeled with Gd(III)-PyMTA and microinjected into Xenopus laevis oocytes. The overtone coefficients are determined to be the same for protonated aqueous solutions and inside cells. As compared to in-cell DEER (double electron–electron resonance, also abbreviated as PELDOR), in-cell RIDME features approximately 5 times larger modulation depth and does not show artificial broadening in the distance distributions due to the effect of pseudosecular terms.

Compaction of RNA Duplexes in the Cell**

“…Early in‐cell PELDOR experiments on nucleic acids [24, 25] and proteins [26] have been hampered by the limited stability of conventional paramagnetic tags in the reducing intracellular environment [27] . This has motivated the development of several classes of reduction‐resistant spin labels based on substituted nitroxides, [27] Gd III chelates [28–31] or trityl radicals, [32–35] for which successful in‐cell distance measurements have been demonstrated [28, 36–48] . Here we chose a nitroxide functional group that is flanked by gem ‐diethyl substituents to sterically shield the spin label from reduction [27, 49–56] .…”

“…[27] This has motivated the development of several classes of reduction-resistant spin labels based on substituted nitroxides, [27] Gd III chelates [28][29][30][31] or trityl radicals, [32][33][34][35] for which successful in-cell distance measurements have been demonstrated. [28,[36][37][38][39][40][41][42][43][44][45][46][47][48] Here we chose a nitroxide functional group that is flanked by gem-diethyl substituents to sterically shield the spin label from reduction. [27,[49][50][51][52][53][54][55][56] Another important criterion in the selection of a spin label for PELDOR experiments is to limit the flexibility of the tether between the radical and the nucleic acid in order to increase the accuracy of the measurements.…”

Site-directed attachment of photoexcitable spin labels for light-induced pulsed dipolar spectroscopy

“…As the applications of pulsed dipolar spectroscopy (PDS) broaden, the repertory of spin labels now comprises not only the routinely used nitroxides 7 but also other paramagnetic species. [8][9][10][11] To accommodate the spectral and relaxational differences between these labels, other techniques than the established double electronelectron resonance (DEER) experiment, 12 such as relaxationinduced dipolar modulation enhancement (RIDME) 13,14 and double-quantum coherence (DQC), 15 are stepping into the spotlight. Several examples have also illustrated the advantages of combining spectroscopically orthogonal spin labels, e.g., Cu(II) and nitroxides.…”

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

“…In the recent years, the introduction of biocompatible spin labels based on gadolinium ions (Gd 3+ ) [27,[87][88][89][90][91][92][93] has led to major progress in the field of in-cell EPR. Spin-labelled recombinantly produced soluble proteins have been already introduced into cells at micromolar concentrations using electroporation methods and interspin distances could be successfully detected (see, e.g., Ref.…”