We report the first example of EPR distance determination using photoexcitable spin labels site-selectively attached via click or thiol chemistry.

No abstract

Distance determination with pulsed EPR has become an important technique for the structural investigation of biomacromolecules, with double electron–electron resonance spectroscopy (DEER) as the most important method. GdIII-based spin labels are one of the most frequently used spin labels for DEER owing to their stability against reduction, high magnetic moment, and absence of orientation selection. A disadvantage of GdIII–GdIII DEER is the low modulation depth due to the broad EPR spectrum of GdIII. Here, we introduce laser-induced magnetic dipole spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique. We show that the excited state of the porphyrin is not disturbed by the presence of the GdIII complex and that herewith modulation depths of almost 40% are possible. This is significantly higher than the value of 7.2% that was achieved with GdIII–GdIII DEER.

Abstract. EPR distance determination in the nanometre region has become an important tool for studying the structure and interaction of macromolecules. Arbitrary waveform generators (AWGs), which have recently become commercially available for EPR spectrometers, have the potential to increase the sensitivity of the most common technique, double electron–electron resonance (DEER, also called PELDOR), as they allow the generation of broadband pulses. There are several families of broadband pulses, which are different in general pulse shape and the parameters that define them. Here, we compare the most common broadband pulses. When broadband pulses lead to a larger modulation depth, they also increase the background decay of the DEER trace. Depending on the dipolar evolution time, this can significantly increase the noise level towards the end of the form factor and limit the potential increase in the modulation-to-noise ratio (MNR). We found asymmetric hyperbolic secant (HS{1,6}) pulses to perform best for short DEER traces, leading to a MNR improvement of up to 86 % compared to rectangular pulses. For longer traces we found symmetric hyperbolic secant (HS{1,1}) pulses to perform best; however, the increase compared to rectangular pulses goes down to 43 %.

Abstract. Laser-induced magnetic dipole (LaserIMD) spectroscopy and light-induced double electron–electron resonance (LiDEER) spectroscopy are important techniques in the emerging field of light-induced pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy (light-induced PDS). These techniques use the photoexcitation of a chromophore to the triplet state and measure its dipolar coupling to a neighboring electron spin, which allows the determination of distance restraints. To date, LaserIMD and LiDEER have been analyzed with software tools that were developed for a pair of two S=1/2 spins and that neglected the zero-field splitting (ZFS) of the excited triplet. Here, we explore the limits of this assumption and show that the ZFS can have a significant effect on the shape of the dipolar trace. For a detailed understanding of the effect of the ZFS, a theoretical description for LaserIMD and LiDEER is derived, taking into account the non-secular terms of the ZFS. Simulations based on this model show that the effect of the ZFS is not that pronounced in LiDEER for experimentally relevant conditions. However, the ZFS leads to an additional decay in the dipolar trace in LaserIMD. This decay is not that pronounced in Q-band but can be quite noticeable for lower magnetic field strengths in X-band. Experimentally recorded LiDEER and LaserIMD data confirm these findings. It is shown that ignoring the ZFS in the data analysis of LaserIMD traces can lead to errors in the obtained modulation depths and background decays. In X-band, it is additionally possible that the obtained distance distribution is plagued by long distance artifacts.