Extending electron paramagnetic resonance to nanoliter volume protein single crystals using a self-resonant microhelix

Abstract

Electron paramagnetic resonance (EPR) spectroscopy on protein single crystals is the ultimate method for determining the electronic structure of paramagnetic intermediates at the active site of an enzyme and relating the magnetic tensor to a molecular structure. However, crystals of dimensions typical for protein crystallography (0.05 to 0.3mm) provide insufficient signal intensity. In this work, we present a microwave self-resonant microhelix for nanoliter samples that can be implemented in a commercial X-band (9.5 GHz) EPR spectrometer. The self-resonant microhelix provides a measured signal-to-noise improvement up to a factor of 28 with respect to commercial EPR resonators. This work opens up the possibility to use advanced EPR techniques for studying protein single crystals of dimensions typical for x-ray crystallography. The technique is demonstrated by EPR experiments on single crystal [FeFe]-hydrogenase (Clostridium pasteurianum; CpI) with dimensions of 0.3 mm by 0.1 mm by 0.1 mm, yielding a proposed g-tensor orientation of the Hox state.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

“…Single-crystal EPR studies have been made to characterize the stable tyrosyl radical in ribonucleotide reductase, photosystem II, and the oxidized state of [FeFe]-hydrogenase by using special sample containers (63)(64)(65). Previous methods used a limited number of crystals, which creates spectra with orientation dependence yielding additional information such as g-tensor parameters.…”

Scalable microresonators for room-temperature detection of electron spin resonance from dilute, sub-nanoliter volume solids

“…Spin detection in volume-limited samples has applications in fields ranging from solid-state physics to structural biology (1)(2)(3)(4). Magnetic resonance spectroscopies based on inductive detection are powerful and versatile techniques that can provide atomic-level structural and functional information for a wide range of samples under broadly variable conditions.…”

Hyperfine spectroscopy in a quantum-limited spectrometer

“…In parallel, recent results have shown that the inductive detection method can also be pushed to much higher absolute sensitivity than previously achieved, using planar microresonators (Narkowicz et al, 2008;Artzi et al, 2015) and micro-helices (Sidabras et al, 2019). Superconducting resonators (Wallace and Silsbee, 1991;Benningshof et al, 2013;Sigillito et al, 2014) are particularly useful in that context since they combine low mode volume and narrow linewidth κ. Inductive-detection spectrometers relying on a superconducting planar micro-resonator combined with a S. Probst et al: Hyperfine spectroscopy in a quantum-limited spectrometer Josephson parametric amplifier (JPA), cooled down to millikelvin temperatures (Bienfait et al, 2015;Eichler et al, 2017;Probst et al, 2017), have achieved a sensitivity of 10 spin/…”