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 a increase in the P(r) maximum from T=6 to 20 mu s. ProteinA has a bimodal P(r) distribution, and the relative height of the shorter distance peak at T=10 mu s, 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.