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 ²³Na signals at two labeled sites in the Na⁺/aspartate symporter Glt_Tk. Comparison of experimental ²³Na 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.