Issue 24, 2022

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

Abstract

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.

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

Supplementary files

Article information

Article type
Paper
Submitted
02 Mar 2022
Accepted
10 May 2022
First published
10 May 2022

Phys. Chem. Chem. Phys., 2022,24, 14727-14739

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

X. Bogetti, Z. Hasanbasri, H. R. Hunter and S. Saxena, Phys. Chem. Chem. Phys., 2022, 24, 14727 DOI: 10.1039/D2CP01032A

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