Dependence of distance distributions derived from double electron-electron resonance pulsed EPR spectroscopy on pulse-sequence time

Angew Chem Int Ed Engl. 2015 Apr 27;54(18):5336-9. doi: 10.1002/anie.201500640. Epub 2015 Mar 10.


Pulsed double electron-electron resonance (DEER) provides pairwise P(r) distance distributions in doubly spin labeled proteins. We report that in protonated proteins, P(r) is dependent on the length of the second echo period T owing to local environmental effects on the spin-label phase memory relaxation time Tm . For the protein ABD, this effect results in a 1.4 Å increase in the P(r) maximum from T=6 to 20 μs. Protein A has a bimodal P(r) distribution, and the relative height of the shorter distance peak at T=10 μs, the shortest value required to obtain a reliable P(r), is reduced by 40 % relative to that found by extrapolation to T=0. Our results indicate that data at a series of T values are essential for quantitative interpretation of DEER to determine the extent of the T dependence and to extrapolate the results to T=0. Complete deuteration (99 %) of the protein was accompanied by a significant increase in Tm and effectively abolished the P(r) dependence on T.

Keywords: EPR spectroscopy; deuteration; distance distributions; phase memory time; proteins.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Deuterium / chemistry
  • Deuterium Exchange Measurement / methods*
  • Electron Spin Resonance Spectroscopy / methods*
  • Models, Chemical
  • Protein Conformation
  • Proteins / chemistry*
  • Spin Labels


  • Proteins
  • Spin Labels
  • Deuterium