Light-induced dipolar spectroscopy – A quantitative comparison between LiDEER and LaserIMD

Abstract

Nanometric distance measurements with EPR spectroscopy yield crucial information on the structure and interactions of macromolecules in complex systems. The range of suitable spin labels for such measurements was recently expanded with a new class of light-inducible labels: the triplet state of porphyrins. Importantly, accurate distance measurements between a triplet label and a nitroxide have been reported with two distinct light-induced spectroscopy techniques, (light-induced) triplet-nitroxide DEER (LiDEER) and laser-induced magnetic dipole spectroscopy (LaserIMD). In this work, we set out to quantitatively compare the two techniques under equivalent conditions at Q band. Since we find that LiDEER using a rectangular pump pulse does not reach the high modulation depth that can be achieved with LaserIMD, we further explore the possibility of improving the LiDEER experiment with chirp inversion pulses. LiDEER employing a broadband pump pulse results in a drastic improvement of the modulation depth. The relative performance of chirp LiDEER and Laser-IMD in terms of modulation-to-noise ratio is found to depend on the dipolar evolution time: While LaserIMD yields higher modulation-to-noise ratios than LiDEER at short dipolar evolution times (τ=2μs), the high phase memory time of the triplet spins causes the situation to revert at τ=6μs.

Site-directed attachment of photoexcitable spin labels for light-induced pulsed dipolar spectroscopy

“…These effects have also been observed in some experimental data. 32,33 Note that experimental LaserIMD traces are typically recorded by moving the Hahn-echo sequence through a fixed laser flash, which results in a mirror inverted trace. As none of the three simulations for the photoexcitable spin labels showed major deviations from those of porphyrin, we concluded that RB, EO and AT12 are suitable labels for LaserIMD measurements.…”

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…The first work in this area was conducted on a peptide-based molecular ruler containing a nitroxide probe and porphyrin moiety. [31][32][33] It was found that the relative signal-to-noise of the two techniques depends strongly on the degree of excitation that can be achieved by the pump pulse used in LiDEER, the laser excitation, the relative relaxation times of the two species being investigated and the inter spin distance range that needs to be probed. [29] The dipolar measurements were performed with light-induced DEER (LiDEER), [25,26] a variation of the conventional 4-pulse DEER sequence where a laser pulse is used to generate the triplet state before the application of dichromatic microwave pulses: the detection frequency is resonant with the photo-induced porphyrin triplet and the pump is resonant with the stable nitroxide radical.…”

“…Additionally, for the first time, the LiRIDME sequence, in the five-pulse dead-time free version, is applied to a triplet probe providing evidence that the longitudinal relaxation properties of the triplet state can be favorable for the application of this technique. [31,32] RIDME has previously been shown to be more sensitive than DEER for measuring inter-spin interactions between paramagnetic species with different longitudinal (T 1 ) relaxation times and in the presence of broad EPR spectra. The ReLaserIMD data set is good, characterized by a modulation depth of 18 % and a signal to noise S/N ' 49.…”

“…[30] Comparison between the two techniques was carried out both at X-band and at Q-band. [31][32][33] It was found that the relative signal-to-noise of the two techniques depends strongly on the degree of excitation that can be achieved by the pump pulse used in LiDEER, the laser excitation, the relative relaxation times of the two species being investigated and the inter spin distance range that needs to be probed. LaserIMD and LiDEER can therefore be seen as complementary to one another.…”

“…Each of the two methods has its own specific factors influencing the value of the modulation depth as previously discussed: it depends on the excitation efficiency of the pump pulse for LiDEER and on the laser excitation and quantum yield for (Re)LaserIMD. [31,32] RIDME has previously been shown to be more sensitive than DEER for measuring inter-spin interactions between paramagnetic species with different longitudinal (T 1 ) relaxation times and in the presence of broad EPR spectra. [39] To this end, LiRIDME (see Figure 3 for pulse sequence) detecting on the nitroxide signal and allowing the broad triplet species to relax was also measured.…”