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

Abstract

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.

Figure 1

Four Pulse DEER.^[1d] The total duration of the second echo period is T (= 2τ₂).

Figure 2

Effect of total second spin-echo period time T on Q-band DEER measurements for ABD and Protein A. The raw DEER echo curves are shown in the top panels (see SI Figure S2 for background subtracted curves) and the P(r) distributions generated by Tikhonov regularization (TR) using DeerAnalysis 2013^[6] are displayed in the middle panels. The lower panels plot the dependence of either the maximum of the P(r) distribution for protonated ABD (bottom left panel) or the ratio of peaks heights for the two maxima of the bimodal P(r) distribution for Protein A on T. (a–c) Protonated ABD domain: T = 6 (black), 8 (red), 10 (green), 12 (blue), 14 (orange), 16 (brown), 18 (turquoise) and 20 μs (violet). The original DeerAnalysis^[6] results were normalized to yield a peak maximum of 1 and are presented in (b). The position of the P(r) maximum was determined by fitting the 7 points around the TR-derived distribution maximum to a quadratic equation and setting the first derivative of this function to zero. The four open circles at T= 10 μs in (c) and (l) represent the results of four different experiments and were included to provided a measure of data reproducibility (SI Figure S4). (d–f) Protonated Protein A: T = 6 (black), 8 (red), 10 (green), 12 (blue), 14 (orange), 16 (brown), 18 (turquoise) and 20 μs (violet). (g–i) ~80% perdeuterated Protein A labeled with MTSL-d₁₅: T = 6 (black), 8 (red), 10 (green), 12 (blue), 15 (dark green), 20 (maroon), 24 (turquoise), 30 (violet), 35 (cyan), 40 (magenta), 50 (orange) and 60 μs (indigo). (k–l) 99% perdeuterated Protein A labeled with MTSL-d₁₅: T = 10 (black), 20 (red), 30 (green), 40 (blue), 50 (orange) and 60 (brown) ms. The original DeerAnalysis TR results for Protein A were normalized to the longer distance peak. The shot repetition times are 3.1 ms for ABD, 3.8 ms for protonated Protein A, and 4.6 ms for perdeuterated (~80% and 99%) Protein A. The T dependence of the ratio of peak heights in the P(r) distribution for Protein A (panels f, i and l for protonated, ~80% perdeuterated and 99% perdeuterated samples, respectively) was fit to either a straight line (black dashes) or in the case of protonated and ~80% perdeuterated Protein A to the ratio of two stretched exponentials (red curves in panels f and i, respectively, see main text). The parameters for the latter are a = 1.2, T_m1 = 9 μs, T_m2 = 13 μs, and x₁=x₂ =1.5 for protonated protein A; and 1.3, 25 μs, 29 μs and 1.25, respectively for ~80% perdeuterated Protein A. These T_m values are close to those obtained from a two-pulse Hahn spin-echo experiment (SI Figure S5). DEER data were collected at Q-band (33.8 GHz) on a Bruker E-580 spectrometer equipped with a 150 W traveling wave tube amplifier and a model ER5107D2 probe. Experiments employed 8 ns pump (ELDOR) π pulses, and 12 ns π/2 and 24 ns π observe pulses, with a 95 MHz frequency difference between pump and observe pulses. The pump frequency was centered at the field spectrum maximum. The τ₁ value for the first echo period time (cf. Figure 1) of 400 ns was incremented 8 times in 16 ns increments to average ²H modulation. The pump pulse was incremented in 16 ns steps. Sample temperature = 48K. The bandwidth of the over-coupled resonator was ~120 MHz. All samples were placed in 1.1 mm internal diameter quartz tubes (Wilmad WG-221T-RB) and flash frozen in liquid N₂. DEER curves were sampled to ~4.4 μs for the various τ₂ delays as collecting more data was not deemed to be a good use of instrument time. A cutoff of 4.3 μs was used to process all data. Cutoffs of 2.3 and 3.3 μs were used for the τ₂ (T/2) = 3 and 4 μs data sets, respectively. Acquisition over the full τ₂ range was not used in the latter cases because of a persistent “2+1” echo perturbation of the DEER curve at a time of ~τ₁ from the final observe π pulse. Total data collection times varied from ~1 to ~22 hrs, with the goal of achieving comparable S/N for all spectra. The pulse gate time used for echo integration was 30–34 ns. The TR parameter α (100 for protein ABD and 10 for Protein A) was determined by examination of the relevant L-curves^[6] (see SI Figure S3). The DeerAnalysis^[6] homogeneous model with dimension = 3 (i.e. exponential background) was used to fit and automatically subtract the background; the resulting Pake patterns indicate good separation of inter- and intramolecular contributions.