title: W-Band pulse EPR distance measurements in peptides using Gd(3+)-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.
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Gordon-Grossman
authors.forename: Ilia
authors.initials: I
authors.lastname: Kaminker
authors.forename: Yana
authors.initials: Y
authors.lastname: Gofman
authors.forename: Yechiel
authors.initials: Y
authors.lastname: Shai
authors.forename: Daniella
authors.initials: D
authors.lastname: Goldfarb
mesh_terms: D056831:Coordination Complexes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005682:Gadolinium / Q000737:chemistry*; D008555:Melitten / Q000737:chemistry; D009010:Monte Carlo Method; D010455:Peptides / Q000737:chemistry*; D010848:Picolinic Acids / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D056831:Coordination Complexes; D010455:Peptides; D010848:Picolinic Acids; D013113:Spin Labels; D008555:Melitten; D005682:Gadolinium; C004860:dipicolinic acid