Rigid and stable nitroxide spin label for high-resolution distance measurements on proteins by DEER experiments

The distance distributions between two site-specifically anchored spin labels in a protein, measured by pulsed electron-electron double resonance (PELDOR or DEER), provide rich sources of structural and conformational restraints on the proteins or their complexes. The rigid connection of the nitroxide spin label to the protein improves the accuracy and precision of distance measurement. We report a new spin labelling approach by formation of thioester bond between nitroxide (NO) spin label, NOAI (NO spin labels activated by acetylimidazole), and a protein thiol, and this spin labeling method has demonstrated high performance in DEER distance measurement on proteins. The results showed that NOAI has shorter connection to the protein ligation site than 2, 2, 5, 5-tetramethyl-pyrroline-1-oxyl methanethiosulfonate (MTSL) and 3-maleimido-proxyl (M-Prox) in the respective protein conjugate and produces narrower distance distributions for the tested proteins including ubiquitin (Ub), immunoglobulin-binding β1 domain of streptococcal protein G (GB1), and second mitochondria-derived activator of caspases (Smac). The NOAI protein conjugate connected by a thioester bond is resistant to reducing reagent and offers high-fidelity DEER distance measurements in cell lysates.