Efficient sampling of molecular orientations for Cu(II)-based DEER on protein labels.

Combining rigid Cu(II) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron-nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu(II)-labeled proteins to evaluate pulse excitation in the context of double electron-electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment and the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations.