Enhancing sensitivity of Double Electron-Electron Resonance (DEER) by using Relaxation-Optimized Acquisition Length Distribution (RELOAD) scheme

Abstract

Over the past decades pulsed electron-electron double resonance (PELDOR), often called double electron-electron resonance (DEER), became one of the major spectroscopic tools for measurements of nanometer-scale distances and distance distributions in non-crystalline biological and chemical systems. The method is based on detecting the amplitude of the primary (3-pulse DEER) or refocused (4-pulse DEER) spin echo for the so-called “observer” spins when the other spins coupled to the former by a dipolar interaction are flipped by a “pump” pulse at another EPR frequency. While the timing of the pump pulse is varied in steps, the positions of the observer pulses are typically fixed. For such a detection scheme the total length of the observer pulse train and the electron spin memory time determine the amplitude of the detected echo signal. Usually, the distance range considerations in DEER experiments dictate the total length of the observer pulse train to exceed the phase memory time by a factor of few and this leads to a dramatic loss of the signal-to-noise ratio (SNR). While the acquisition of the DEER signal seems to be irrational under such conditions, it is currently the preferred way to conduct DEER because of an effective filtering out of all other unwanted interactions. Here we propose a novel albeit simple approach to improve DEER sensitivity and decrease data acquisition time by introducing the signal acquisition scheme based on RELaxation Optimized Acquisition (Length) Distribution (DEER-RELOAD). In DEER-RELOAD the dipolar phase evolution signal is acquired in multiple segments in which the observer pulses are fixed at the positions to optimize SNR just for that specific segment. The length of the segment is chosen to maximize the signal acquisition efficiency according the phase relaxation properties of the spin system. The total DEER trace is then obtained by “stitching” the multiple segments into a one continuous trace. The utility of the DEER-RELOAD acquisition scheme has been demonstrated on an example of the standard 4-pulse DEER sequence applied to two membrane protein complexes labeled with nitroxides. While theoretical gains from the DEER-RELOAD scheme increase with the number of stitched segments, in practice, even dividing the acquisition of the DEER trace into two segments may improve SNR by a factor of >3, as it has been demonstrated for one of these two membrane proteins.

Non-uniform sampling in pulse dipolar spectroscopy by EPR: the redistribution of noise and the optimization of data acquisition

“…The second method is RELOAD (Relaxation-Optimized Acquisition Length Distribution), where the dipolar trace is measured segmentally, and the segments are stitched into a full trace. 44 RELOAD can be implemented as a NUA scheme. Both schemes gave enhanced accuracy and reduced measuring time for a complicated P(r).…”

Optimising broadband pulses for DEER depends on concentration and distance range of interest

“…In the last few years DEER (double electron-electron resonance) has developed into an important technique for the determination of distances in the nanometre range (Jeschke, 2012;Milov et al, 1981Milov et al, , 1984 and in particular into a suitable tool for studying biological macromolecules (e.g. proteins (Jeschke, 2012;Robotta et al, 2014) or RNA/DNA (Grytz et al, 2017;Kuzhelev et al, 2018)).…”

Optimising broadband pulses for DEER depends on concentration and distance range of interest

“…Increasing the sensitivity of DEER spectroscopy is an active field of research (Borbat et al, 2013;Breitgoff et al, 2017;Doll et al, 2015;Jeschke et al, 2004;Lovett et al, 2012;Milikisiyants et al, 2019;Polyhach et al, 2012;Tait and Stoll, 2016;Teucher and Bordignon, 2018). A very elegant approach to increasing DEER sensitivity has been made possible by the availability of arbitrary waveform generators with time resolution in the nanosecond region as they allow the generation of 30 broadband microwave pulses (Doll et al, 2013;Doll and Jeschke, 2017;Spindler et al, 2017).…”