W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity

Abstract

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 μs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.

Distance measurements between manganese(ii) and nitroxide spin-labels by DEER determine a binding site of Mn2+ in the HP92 loop of ribosomal RNA

“…3c. 40 In combining the NO-NO distances obtained from RNA (3,31) with those obtained from the Mn-NO results we assumed that addition of the equimolar amount of Mn 2+ did not alter the structure of the RNA significantly. 3d).…”

Gd(iii)–Gd(iii) EPR distance measurements – the range of accessible distances and the impact of zero field splitting

“…59 It has recently been shown that Gd(III)-nitroxyl DEER measurements at the Q-and W band is yet another attractive approach for distance determination. 38,39,41 In this case, the effect of the pseudo-secular term should pose less of a problem, because the overlap between the nitroxyl radical transition and the Gd(III) transitions is limited to the broad background of the Gd(III) spectrum, and is attributed to transitions other than the central one. A theoretical analysis that takes into account the ZFS for such measurements has been reported.…”

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…Modifications of this approach involve the use of non-identical spin labels, in particular a Gd(III) center and a nitroxide [3,4], as well as different experimental schemes, such as relaxation-induced dipolar modulation (RIDME) [5] or continuous-wave EPR [6]. Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23].…”