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.

Distance measurements using double electron–electron resonance (DEER) and Gd3+ chelates for spin labels (GdSL) have been shown to be an attractive alternative to nitroxide spin labels at W-band (95 GHz). The maximal distance that can be accessed by DEER measurements and the sensitivity of such measurements strongly depends on the phase relaxation of Gd3+ chelates in frozen, glassy solutions. In this work, we explore the phase relaxation of Gd3+-DOTA as a representative of GdSL in temperature and concentration ranges typically used for W-band DEER measurements. We observed that in addition to the usual mechanisms of phase relaxation known for nitroxide based spin labels, GdSL are subjected to an additional phase relaxation mechanism that features an increase in the relaxation rate from the center to the periphery of the EPR spectrum. Since the EPR spectrum of GdSL is the sum of subspectra of the individual EPR transitions, we attribute this field dependence to transition dependent phase relaxation. Using simulations of the EPR spectra and its decomposition into the individual transition subspectra, we isolated the phase relaxation of each transition and found that its rate increases with |ms|. We suggest that this mechanism is due to transient zero field splitting (tZFS), where its magnitude and correlation time are scaled down and distributed as compared with similar situations in liquids. This tZFS induced phase relaxation mechanism becomes dominant (or at least significant) when all other well-known phase relaxation mechanisms, such as spectral diffusion caused by nuclear spin diffusion, instantaneous and electron spin spectral diffusion, are significantly suppressed by matrix deuteration and low concentration, and when the temperature is sufficiently low to disable spin lattice interaction as a source of phase relaxation.

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron‐electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide‐DOTA‐Gd(III) in the N‐ and C‐terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo‐CaM and holo‐CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo‐ and holo‐CaM bind to target proteins in a similar way as apo‐ and holo‐CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in‐cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in‐cell components. These results show‐case the importance of in‐cell analyses of protein structures.

Correction for 'Gd(iii)-Gd(iii) EPR distance measurements - the range of accessible distances and the impact of zero field splitting' by Arina Dalaloyan et al., Phys. Chem. Chem. Phys., 2015, 17, 18464-18476.

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.