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

Abstract

Gd(III) complexes have emerged as spin labels for distance determination in biomolecules through double-electron-electron resonance (DEER) measurements at high fields. For data analysis, the standard approach developed for a pair of weakly coupled spins with S = 1/2 was applied, ignoring the actual properties of Gd(III) ions, i.e. S = 7/2 and ZFS (zero field splitting) ≠ 0. The present study reports on a careful investigation on the consequences of this approach, together with the range of distances accessible by DEER with Gd(III) complexes as spin labels. The experiments were performed on a series of specifically designed and synthesized Gd-rulers (Gd-PyMTA-spacer-Gd-PyMTA) covering Gd-Gd distances of 2-8 nm. These were dissolved in D2O-glycerol-d8 (0.03-0.10 mM solutions) which is the solvent used for the corresponding experiments on biomolecules. Q- and W-band DEER measurements, followed by data analysis using the standard data analysis approach, used for S = 1/2 pairs gave the distance-distribution curves, of which the absolute maxima agreed very well with the expected distances. However, in the case of the short distances of 2.1 and 2.9 nm, the distance distributions revealed additional peaks. These are a consequence of neglecting the pseudo-secular term in the dipolar Hamiltonian during the data analysis, as is outlined in a theoretical treatment. At distances of 3.4 nm and above, disregarding the pseudo-secular term leads to a broadening of a maximum of 0.4 nm of the distance-distribution curves at half height. Overall, the distances of up to 8.3 nm were determined, and the long evolution time of 16 μs at 10 K indicates that a distance of up to 9.4 nm can be accessed. A large distribution of the ZFS parameter, D, as is found for most Gd(III) complexes in a frozen solution, is crucial for the application of Gd(III) complexes as spin labels for distance determination via Gd(III)-Gd(III) DEER, especially for short distances. The larger ZFS of Gd-PyMTA, in comparison to that of Gd-DOTA, makes Gd-PyMTA a better label for short distances.

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

“…The deviation is up to 1.6% and presumably due to approximations in distance prediction and experimental uncertainty. In comparison to recent experiments at W-band [20], we notice apparent differences for the long Gdruler 1 11 . These are discussed in Section 6.3 of the SI.…”

“…Moreover, the frequency spacing f 2 between pump and observation at optimum η 2P is larger than for a single pump pulse. In this way, proximity problems such as residual excitation of the A spins by the pump pulse or potential deviations from the weak coupling limit of the dipole interaction [7,20] are alleviated. Another point worth mentioning is that there is no significant overhead in setting up the experiment with consecutive chirps.…”

“…Besides possible distortions due to the duration of chirp pulses, the ultra-wideband frequency windows of the pump pulses may influence the obtained distance information. While orientation selection effects in Gd-Gd distance measurements are not significant [1], contributions of the ZFS may be altered [7,20]. For further reference, an overview over the subtle differences in distance distributions obtained with Gd-ruler 1 3 is provided in Fig.…”

“…The dipolar evolutions windows used here allow to extract mean distances as well as the width of the distance distribution [46]. Mean distances predicted by taking the bending of the spacer into account [20] are shown by the blue vertical lines underneath the distance peaks (see Section 6.4 of the SI). Mean distances extracted from the experimental distributions (see caption and Section 6.3 of the SI) are slightly longer than the predicted distances.…”

“…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].…”