Investigating membrane protein conformational dynamics and noncovalent interactions via electron paramagnetic resonance spectroscopy.

The advent of machine learning structural prediction tools has largely accelerated bottom-up structural biology and provides static Anfinsenian models with near-experimental accuracy, complementing X-ray crystallography and cryogenic electron microscopy. However, these techniques often overlook two fundamental aspects of biomolecular structure-function relationships: (i) conformational equilibria and (ii) transient noncovalent interactions. Indeed, these considerations are particularly important for integral membrane proteins (MPs), frequently serving mechanistic roles, such as conformational plasticity in ion channels facilitating heterogeneous state transitions, or dynamic binding partners modulating signal transduction in receptors and small-molecule transporters. In this vein, electron paramagnetic resonance (EPR) spectroscopy, an ensemble method, provides a powerful toolbox to guide high-resolution structural data by directly reporting on conformational subensembles and noncovalent binding events. Herein, we highlight recent EPR applications to integral MPs and emerging synergies with bottom-up atomistic approaches to enhance insights into structure-dynamics-function relationships.