Spins Afar─Long-Range Pulse Dipolar EPR Spectroscopy Uncovers Curvature in TRIM25’s Coiled Coil

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy (PDS) is a powerful method for determining nanometer distances in biomolecular systems. This methodology has been successfully used to validate structure hypotheses and to identify disorder. However, inherent distance limitations are under-explored. Many challenging biomolecular applications require long distance information, driving the need for a benchmarking study. We demonstrate distances up to 10.5 nm in TRIM25 reliably extracted from data obtained under standard sample conditions. By protein perdeuteration accurate distance measurements up to 17.3 nm were enabled. Enhancing sensitivity through optimization of the measurement and sample preparation conditions the achievable dipolar evolution time was extended to 150 μs. Thus, distances close to 20 nm should become experimentally accessible, highlighting the potential of advanced PDS methodologies for probing previously inaccessible long-range structural information in complex systems.