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In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors.

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However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s.

The range of applications of DEER include evaluation of static structures in solution (e.g., Zhou et al., 2005) or native-like environments (e.g., Barrett et al., 2012), probing ligand-induced conformational changes, characterization of equilibrium fluctuations, and investigation of protein-protein interactions (Hilger et al., 2005 Hilger et al., 2007 Kim et al., 2011 Edwards et al., 2014). In the protein space, unique insight was revealed on ion channel architecture and gating (Pliotas et al., 2012 Dellisanti et al., 2013 Dalmas et al., 2014 Dürr et al., 2014 Puljung et al., 2014 Raghuraman et al., 2014 Arrigoni et al., 2016 Zhu et al., 2016 Evans et al., 2020), transporter alternating access and energy landscapes (Claxton et al., 2010 Mittal et al., 2012 Georgieva et al., 2013 Kazmier et al., 2014a Kazmier et al., 2014b Masureel et al., 2014 Joseph et al., 2015 Dastvan et al., 2016 Martens et al., 2016 Timachi et al., 2017 Verhalen et al., 2017 Göddeke et al., 2018 Dastvan et al., 2019 Joseph et al., 2019 Nyenhuis et al., 2020), and receptor activation and allosteric modulation (Park et al., 2006 Kim et al., 2012 Kang et al., 2015 Yee et al., 2015 Van Eps et al., 2017 Van Eps et al., 2018 Wingler et al., 2019 Elgeti and Hubbell, 2021). DEER has been applied to multiple proteins, RNA, and DNA systems. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg ²⁺ but that conducting states become more populated in Mg ²⁺ -free conditions. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg ²⁺. We find that neither the Mg ²⁺ -bound closed structure nor the Mg ²⁺ -free open forms are sufficient to explain the average conformation of CorA. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg ²⁺ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation.