DEER is also known as pulsed electron double resonance (PELDOR). DEER has been a widely used biophysical technique for measuring distances between two spin labels on membrane proteins in the range of 18–60 Å
[124][125][126]. In DEER spectroscopy, a dipolar coupling between two spins is measured by monitoring one set of spins while exciting another set of spins with a second microwave frequency, leading to the measurement of the distance between them
[127][124][128]. Nitroxide spin labeling based DEER spectroscopy is very popular for investigating the secondary, tertiary, and quaternary structures and conformational dynamics of a wide variety of macromolecules
[19][40][53][57][116][117][118][119][120][121][122][123][124][125][126][129][130][131][132][133][134]. In addition to nitroxide spin labels, other spin labels such as functionalized chelators of paramagnetic lanthanides (Gd
III), carbon-based radicals (trityl), and metals such as copper (Cu
II) have been recently utilized for DEER measurements on membrane proteins
[52][135][136][137][138]. There is also a disadvantage to using non-nitroxide spin labels. The Gd-based and trityl labels are bulkier than nitrixide spin labels, which can cause perturbation in protein structure and function
[52]. Hence, care must be taken while choosing spin-labeling sites to avoid these perturbations. shows the DEER distance measurement method used for studying membrane proteins. The dipolar coupling frequency (ν
12) is inversely related to the third power of the distance between two spin labels (ν
12 ∝
1/d3)
[127][139]. The most commonly used four pulse DEER sequence is shown in B. In the four pulse DEER sequence, an echo is generated by applying three microwave pulses with specific positions to the one set of spins S
1 at the probe frequency ν
1. Another set of spins, S
2, is flipped by applying a 4th pump microwave pulse at varying positions between the last two probe pulses at the frequency ν
2. Consequently, the sign of the dipolar interaction and the amplitude of spin echo change result in the modulation of the echo amplitude as a function of the position of the pump pulse. The forward five-pulse and the reverse five-pulse DEER sequences are shown in C,D, respectively. In the five-pulse DEER sequence (C,D), similar lengths of the inter-pulse delays are applied. This minimizes the effect of the spin diffusion on relaxation, leading to the increase in the refocused echo intensity when compared to that of the four-pulse DEER sequence. The additional pump pulse also helps extend the dipolar evolution window
[140][141][142]. A seven-pulse Carr-Purcell PELDOR sequence with multiple pump pulses (see E) also leads to improved sensitivity in the measurement of long-range distances.
[140][141][143]. These multipulse DEER experiments introduce echo crossing artifacts in DEER traces
[140][141][143]. These artifacts can be minimized by using eight to thirty two-step phase cycling schemes
[140].
Figure 7. Double electron electron resonance DEER spectroscopic method used to measure distances between nitroxide spin labels. (
A) Distance vector (d) between spin S
1 and S
2 on KCNE1 membrane protein (PDB ID: 2k21) at an angle θ with the magnetic field B
0. (
B) Four-pulse DEER sequence. (
C) Forward five-pulse DEER sequence. (
D) Reverse five-pulse DEER sequence. (
E) Seven-pulse CP-PELDOR sequence
[140][141].