Baber James L, Louis John M, Clore G Marius
National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520 (USA).
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.
脉冲双电子-电子共振（DEER）可提供双自旋标记蛋白质中的成对P(r)距离分布。我们报告称，在质子化蛋白质中，由于局部环境对自旋标记相位记忆弛豫时间Tm的影响，P(r)取决于第二个回波周期T的长度。对于蛋白质ABD，这种效应导致P(r)最大值从T = 6 μs增加到20 μs时增加了1.4 Å。蛋白质A具有双峰P(r)分布，在T = 1 μs（获得可靠P(r)所需的最短值）时，较短距离峰的相对高度相对于外推到T = 0时发现的值降低了40%。我们的结果表明，一系列T值的数据对于DEER的定量解释至关重要，以确定T依赖性的程度并将结果外推到T = 0。蛋白质的完全氘化（99%）伴随着Tm的显著增加，并有效地消除了P(r)对T的依赖性。
