Dependence of Distance Distributions Derived from Double Electron–Electron Resonance Pulsed EPR Spectroscopy on Pulse‐Sequence Time

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 T m . 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 T m and effectively abolished the P( r ) dependence on T .