W-Band pulse EPR distance measurements in peptides using Gd 3+-dipicolinic acid derivatives as spin labels

Michal Gordon-Grossman, Ilia Kaminker, Yana Gofman, Yechiel Shai, Daniella Goldfarb*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


We present high field DEER (double electron-electron resonance) distance measurements using Gd3+ (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd3+ 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. Gd3+ 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-Gd3+ labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd3+ 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 [Gd3+]/[Tag] ratio is crucial because excess Gd3+ masked the DEER effect and too little Gd3+ resulted in the formation of Gd3+-tag2 complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd3+ 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 Gd3+ 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.

Original languageEnglish
Pages (from-to)10771-10780
Number of pages10
JournalPhysical Chemistry Chemical Physics
Issue number22
StatePublished - 14 Jun 2011
Externally publishedYes


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