Improved Sensitivity for Long-Distance Measurements in Biomolecules: Five-Pulse Double Electron-Electron Resonance

Abstract

We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron-electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both.

Figure 1

(a) Standard four-pulse DEER sequence with the respective dipolar modulation pattern plotted in green. (b) The four-pulse sequence modified for t₂ = 2t₁ ≡ 2τ to minimize nuclear spin diffusion, thus, increasing the signal, but this shifts the dipolar modulation (in blue) to the middle of the second interval, thereby losing half of the dipolar modulation because the halves are identical. (c) Placing the second pump pulse, 5, after the pulse 4 shifts dipolar modulation toward pulse 4, thereby recovering the full time span, 2τ. The dipolar modulation (green) is reversed in time compared with panel a. Pulse 5 could also be placed at position 5′ before pulse 2, reversing the modulated time trace. Note that time period, t_m, available for recording the dipolar signal is (a) t₂ and (b,c) 2τ.

Figure 2

Comparison of the echo amplitude for the same sample of 40 μM T4L 8/44, recorded as a function of dipolar evolution time period, t_m, with standard four-pulse DEER (in blue) and five-pulse DEER (in red) (cf. Figure 1). The five-pulse DEER signal decays much slower as the pulse sequence expands in time, leading to an increase in the time period, t_max, available for recording the dipolar signal factor by the factor of 1.83 for H₂O (a) and 1.4 for D₂O (b). The echo amplitude shown is in mV and is the receiver output for constant gain.

Figure 3

(a) DEER-5 signal recorded in D₂O buffer is shown after subtracting the residual unwanted DEER-4 type dipolar signal. (b) After standard baseline removal, the DEER-5 data (in black) are plotted together with standard four-pulse DEER data (in red), normalized according to the literature.

Figure 4

Isolation of pure DEER-5 signal. The reference, A, was recorded in the absence of the fifth pulse and used in the removal of the incompletely suppressed dipolar signal of A-type from the raw signal, B, recorded in the presence of the fifth pulse. C indicates the pure five-pulse dipolar signal produced by subtracting scaled down A from B. A–C are normalized to unit amplitude at their maxima, with C shifted by −0.1 for clarity.