W-Band pulse EPR distance measurements in peptides using Gd3+–dipicolinic acid derivatives as spin labels

Abstract

We present high field DEER (double electron-electron resonance) distance measurements using Gd(3+) (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd(3+) spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd(3+) was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd(3+) labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd(3+) labeled peptide we explored the effect of the solution molar ratio of Gd(3+) and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd(3+)]/[Tag] ratio is crucial because excess Gd(3+) masked the DEER effect and too little Gd(3+) resulted in the formation of Gd(3+)-tag(2) complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd(3+) concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd(3+) ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

Nanometer-Range Distance Measurement in a Protein Using Mn2+ Tags

“…The T 1 value of the Mn 2+ -EDTA-tagged p75DD at 10 K was 150 μs, similar to that found for Gd 3+ -labeled proteins (∼100 μs), 13,26 and the T M value was 6.8 μs ( Figure S3, Supporting Information), that is, similar to the T M of 7.6 μs measured for a Gd…”

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

“…Our investigations made so far could not fully elucidate the reason for the reduction of the modulation depth at prolonged τ 2 delays and further experiments are required. A relaxation driven mechanism appears most plausible, as also suggested by the temperature dependence of this effect [10]. Moreover, we expect this effect to be specific to high-spin Gd(III) ions, as we are not aware of such a reduction effect in distance measurements between nitroxides.…”

“…Accordingly, prolongation of the observation window results in a reduction of the modulation depth. In a previous study, it has been shown that this reduction mechanism does not only depend on τ 2 , but also on temperature [10]. In order to gain further insight into the origin of this effect, we studied the dependence on τ 2 of both λ 2P and the echo signal V 0 /V ref at various concentrations.…”

“…Given the large frequency separation between pumped and observed spins, one would expect a majority of these additional evolution pathways to be negligible. Another mechanism is related to stochastic flips of the pumped spins by longitudinal relaxation (see also [10]). With the short longitudinal relaxation times encountered in Gd(III) complexes, one would actually expect such a τ 2 -dependent dephasing mechanism to be relevant.…”

“…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].…”