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Protocol for preparing and characterizing samples for combined microsecond freeze-hyperquenching and electron paramagnetic resonance spectroscopy.

STAR protocols

Authors: Joshua L Wort, Tobias Hett, Adrian Haardt, Olivia Kendall, Hamed Alaei, Olav Schiemann, U Benjamin Kaupp

Microsecond freeze-hyperquenching (MHQ) coupled with pulsed electron-electron double resonance spectroscopy (PELDOR) can resolve conformational changes in biomolecules over space and time. Here, we present a protocol for preparing MHQ samples of a cyclic nucleotide-binding domain by rapid mixing with cyclic adenosine monophosphate. Further, we outline the acquisition and analysis of PELDOR data on MHQ samples and study the dynamics of protein-ligand interactions. This protocol can also be extended to other freeze-quench techniques beyond MHQ and other biomolecules. For complete details on the use and execution of this protocol, please refer to Hett et al..

Copyright © 2026 The Author(s). Published by Elsevier Inc. All rights reserved.

PMID: 41762653

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