Biochemistry, University of Toronto

Electron Paramagnetic Resonance (EPR) spectroscopy is a magnetic resonance technique, similar to Nuclear Magnetic Resonance (NMR) spectroscopy, that is capable of detecting unpaired electrons, or stabilized radicals in a sample. Each of those paramagnetic species have their own individual spectroscopic fingerprint and therefore they can be reliably identified and characterized concerning their molecular frameworks, chemical environment and dynamics in solids or in solution. Historically, EPR facilities are often housed in biochemistry departments for structural and dynamic investigations on macromolecules and (bio)inorganic complexes as the time and length scales to be investigated perfectly fit the capabilities of the available instruments.

However, the vast majority of biological macromolecules don’t bear any unpaired electrons or metal ions and are therefore diamagnetic. Such proteins can be made accessible to EPR spectroscopy via site-directed spin labeling (SDSL). This means that stabilized paramagnetic centres are covalently linked to proteins at topologically selected amino acid residues. There is a multitude of approaches, but the most common SDSL strategy is based on cysteine substitution mutagenesis and targeted reaction of the unique cysteine with a sulfhydryl-specific nitroxide spin label (NO* radical) to introduce a disulfide-linked paramagnetic side chain.

Such SDSL EPR experiments can be used to reveal substantial biophysical information about a protein in terms of site-specific mobility, solvent accessibility and polarity. This helps in identifying, e.g. loop and helix features or their mutual interconversion with time. EPR can also precisely measure distances between two spin-labeled side chains. Most importantly, those distances allow us to unearth unique structural details about a protein adopting its different conformations and dynamics, e.g. upon binding to a substrate molecule (ligand). It can be also investigated how singly labeled proteins align or oligomerize into supramolecular complexes. This means, we can actually observe the proteins or protein complexes at work from a coarse-grained perspective of localized electron spins, very much alike a motion capture.

In principle, EPR experiments can be performed in two different ways:

Continuous Wave (CW) EPR Spectroscopy

With CW EPR Spectroscopy we can measure the microwave absorption properties of paramagnetic centers, thereby yielding information about the electron’s molecular environment, its mobility and solvent accessibility. Moreover, CW EPR can be used to extract the fraction of bound and unbound paramagnetic substrates in solution. This allows us to determine e.g. binding affinities (K_D) of a spin-labeled ligand (so-called spin probes).

Pulsed EPR Spectroscopy

Pulsed EPR Spectroscopy provides a much more intricate approach of investigating paramagnetic species. By choosing microwave pulses of defined strength and duration at a given sample temperature, we can force electron spins to move into any direction we want. We then record how fast they are moving and what kind of interactions they encounter during those processes. This gives us a very unique picture about interactions with surrounding atoms as well as electrons. In particular, Pulsed Dipolar Spectroscopy (PDS), which comprises single and multifrequency techniques, provides us with a variety of tools and experiments to precisely detect distances between spin-labeled sites in a broad range from 18 – 150 Å, however, only when samples are perdeuterated. Double electron-electron resonance (DEER) spectroscopy is generally considered as the gold standard in PDS and is routinely applied in our lab as an integrative structural biology tool. Typically, 20 – 30 µL of a nitroxide labeled protein sample at a concentration of 10 – 200 µM is sufficient to load both, the CW EPR and pulse EPR machine, for achieving highest quality results.

Results from CW EPR and DEER Spectroscopy applied to a spin-labeled protein.

Jessica Besaw and Joerg Reichenwallner discussing DEER data.

Equipment

BRUKER ELEXSYS E500 – CW EPR Spectrometer (X-Band)

Standard Instrument for routine CW EPR applications, sample control, dynamic studies, and sample identification. Data are typically recorded as a field sweep or in a time course experiment to detect microwave absorption, or changes therein (Frequency = 9.7 GHz, Magnetic Field = 0.34 T).

Ned van Eps running a CW EPR Experiment on the E500 machine (ca. 2019).

BRUKER ELEXSYS E580 – Pulse EPR Spectrometer (Q-Band)

High performance and high sensitivity instrument for single and multifrequency pulse EPR applications. Data acquisition can be highly customized. Standard experiments comprise field swept electron spin echo (FS ESE), electron spin echo envelope modulation (ESEEM), inversion recovery, relaxation induced dipolar modulation enhancement (RIDME) and DEER (Frequency = 33.6 GHz, Magnetic Field = 1.2 T). In 2023, the E580 was upgraded with a sustainable Helium closed-cycle cryostat which can reach temperatures down to 7 K without any need for sparse resources as liquid Helium.

Both instruments are currently taken care of by a postdoctoral fellow with more than a decade of experience in running and maintaining such machines.