Insights from Austin

Advancements in science are often spurred by the ‘new.’ Be it new technology, new techniques, or new ideas, novel abilities will beget novel outcomes. The field of EPR has seen this effect throughout its history – beginning with the discovery of the phenomenon itself, and driven forward by breakthroughs such as the development of site-directed spin labeling and distance measurement techniques that enable structural studies on biomolecules; membrane mimetics and in-cell/in-vivo methods to capture physiologically relevant data; in-silico modeling, simulation, and analyses packages; and improvements in hardware, resonators, and pulse-shaping. It is a never-ending upward spiral of new discoveries prompting more questions, requiring new tools to investigate. During my recent visit to the Rocky Mountain Conference on Magnetic Resonance in Denver, several talks emphasized just how far this field has come, how it has benefitted from such advancements, and what new tools could push it even further.

Previously I provided a high-level view of the fundamentals of EPR-based distance measurements. Today, and in future iterations, I will dig deeper into those distance methods, focusing on specific techniques, their strengths, drawbacks, and considerations. Each instance produces similar results – a distance distribution between two unpaired electron spins – but differ in implementation and optimization. Understanding the various factors at play within these methods is crucial to pair the optimal experiment with a given sample and environment.

Many biomolecules of interest lack inherent paramagnetic sites and are thus natively invisible to EPR. To make such systems EPR-detectable, there have been significant and concentrated efforts to develop means by which to incorporate paramagnetic species, either stable organic radicals or paramagnetic metal ions, site-specifically into proteins and nucleic acids. While spin labeling is a nuanced field, I will be introducing the basics of the technique and the three main classes of labels employed today: nitroxides, metal ions, and trityls.

In my first Insights post, wherein I reflected on the Biophysical Society Meeting, I aimed to raise EPR into the common scientific vernacular akin to techniques like CryoEM, which have seen monumental growth and adoption in recent years. I look forward to a time when EPR discussions needn’t start with, ‘Do you know what EPR is?’ However, until that day comes, it may behoove me to introduce you to the technique, providing valuable context and frame of reference for any past and future entries in this series. And so, in this post I will provide a brief overview of EPR, and more specifically, EPR-based distance measurements.

Science is often about context. An outlier can only be determined in the context of the whole distribution. Experimental data is relevant only in the context of its setup and parameters. Biomolecules adopt different behaviors in the context of their local environment. Similarly, my previous Insights have examined EPR in the context of the broad biophysical (BPS) and the narrower EPR (RSC) communities. Most recently I attended Drug Discovery Chemistry 2023, and accordingly, this entry will be examining EPR in the context of the Drug Discovery workflow. Throughout the many excellent talks and presentations, a few key notes were played on refrain – notes that may sound good on an EPR instrument.

In my inaugural post of Insights, I took a bird’s-eye view of how EPR can fit into the broad and diverse landscape of biophysics – an apt reflection after a meeting of the Biophysical Society. This past week I ventured to Leeds to join the Royal Society of Chemistry’s ESR meeting (EPR and ESR are interchangeable) and for a meeting of such focused subject matter, this reflection will appropriately take a more focused approach.

Despite being still an incredibly niche technique, the current state of EPR is something to be excited about. EPR – electron paramagnetic resonance – is a diverse and robust technique that can unlock biophysical information by measuring nanoscale intramolecular distances, determining localized dynamics, and probing paramagnetic binding environments. Every year talented investigators are using this spectroscopy to discover new information and solve old problems. I had the chance to interact with several such groups and see firsthand the new directions the field is heading.