Detection of bound Na ⁺ in a membrane protein by pulse  EPR

Na ⁺ ions generate electron density signals similar to water, making their detection in membrane protein crystal structures challenging. Combined electron spin echo envelope modulation (ESEEM) spectroscopy with site‐directed spin labeling revealed ²³ Na signals at two labeled sites in the Na ⁺ /aspartate symporter Glt _Tk . Comparison of experimental ²³ Na nuclear quadrupole parameters with density functional theory calculations required a single, physically motivated torsional adjustment to improve Na ⁺ coordination. The nitroxide–Na ⁺ distances derived from molecular dynamics simulations and the rotamer analysis of the spin labels, based on a crystal structure, aligned with the experimental values within an uncertainty of 1.5 Å. We conclude that ESEEM is an effective method for localizing and studying the coordination of protein‐bound Na ⁺ ions.