An optimal acquisition scheme for Q-band EPR distance measurements using Cu2+-based protein labels

Recent advances in site-directed Cu2+ labeling of proteins and nucleic acids have added an attractive new methodology to measure the structure-function relationship in biomolecules. Despite the promise, accessing the higher sensitivity of Q-band Double Electron Electron Resonance (DEER) has been challenging for Cu2+ labels designed for proteins. Q-band DEER experiments on this label typically require many measurements at different magnetic fields, since the pulses can excite only a few orientations at a given magnetic field. Herein, we analyze such orientational effects through simulations and show that three DEER measurements, at strategically selected magnetic fields, are generally sufficient to acquire an orientational-averaged DEER time trace for this spin label at Q-band. The modeling results are experimentally verified on Cu2+ labeled human glutathione S-transferase (hGSTA1-1). The DEER distance distribution measured at the Q-band shows good agreement with the distance distribution sampled by molecular dynamics (MD) simulations and X-band experiments. The concordance of MD sampled distances and experimentally measured distances adds growing evidence that MD simulations can accurately predict distances for the Cu2+ labels, which remains a key bottleneck for the commonly used nitroxide label. In all, this minimal collection scheme reduces data collection time by as much as six-fold and is generally applicable to many octahedrally coordinated Cu2+ systems. Furthermore, the concepts presented here may be applied to other metals and pulsed EPR experiments.

Automated summary

Recent advances in site-directed Cu2+ labeling of proteins and nucleic acids have provided a new methodology for measuring the structure-function relationship in biomolecules. However, accessing the higher sensitivity of Q-band DEER has been challenging for Cu2+ labels designed for proteins. In this study, the orientational effects of the label are analyzed through simulations, and it is shown that three strategically selected magnetic field DEER measurements are generally sufficient for obtaining an orientational-averaged DEER time trace at Q-band.