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.
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