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

## TL;DR

This paper provides a detailed protocol for using microsecond freeze-hyperquenching and EPR spectroscopy to study biomolecular conformational changes.

## Contribution

A new protocol for sample preparation and data analysis in MHQ-PELDOR experiments is introduced.

## Key findings

- The protocol enables spatiotemporal resolution of protein dynamics.
- Kinetic parameters can be extracted from freeze-quench pulse dipolar spectroscopy data.
- The method is extendable to other freeze-quench techniques and biomolecules.

## Abstract

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.1

•Optimization of microsecond freeze-hyperquenching sample preparation•Spatiotemporal resolution of protein dynamics by double electron-electron resonance•Extraction of kinetic parameters from freeze-quench pulse dipolar spectroscopy data•Procedures for microsecond freeze-hyperquench device handling and ease of use

Optimization of microsecond freeze-hyperquenching sample preparation

Spatiotemporal resolution of protein dynamics by double electron-electron resonance

Extraction of kinetic parameters from freeze-quench pulse dipolar spectroscopy data

Procedures for microsecond freeze-hyperquench device handling and ease of use

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.