Enabling structural biological electron paramagnetic resonance spectroscopy in membrane proteins through spin labelling

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS), combined with site-directed spin-labelling, represents a powerful tool for the investigation of biomacromolecules, emerging as a keystone approach in structural biology. Increasingly, PDS is applied to study highly complex integral membrane protein systems, such as mechanosensitive ion channels, transporters, G-protein coupled receptors, ion pumps, and outer membrane proteins elucidating their dynamics and revealing conformational ensembles. Indeed, PDS offers a platform to study intermediate or lowly-populated states that are otherwise invisible to other modern methods, such as X-ray crystallography, cryo-EM, and hydrogen-deuterium exchange-mass spectrometry. Importantly, advances in spin labelling strategies welcome a new era of membrane protein investigation under near-native or in-cell conditions. Here, we review recent integral membrane protein PDS applications, and highlight well-suited, emerging spin labelling strategies that show promise for future studies.