SNAr-Based Labeling of Proteins with Trityl Radicals Enables High-Precision, High-Sensitivity, and Long-Range Distance Measurement

Distance measurement using pulsed dipolar electron paramagnetic resonance spectroscopy (PD-EPR) coupled with site-directed spin labeling is a powerful approach to study the structure and dynamics of biomolecules in nearly native environments. However, the accuracy and sensitivity of the distance measurement in cellular systems is often limited by long, flexible, and/or reduction-sensitive linker of spin labels and their low biostability. Herein, we report the first class of aryl-linked trityl spin labels in which pyrimidine and pyridine methylsulfones (FPS1-2) act as protein tagging groups and are directly conjugated to the trityl moiety. FPS1 underwent a specific nucleophilic aromatic substitution (SNAr) reaction with cysteine (Cys) as its free form or in glutathione (GSH) and proteins to produce stable C-S conjugation. The resulting conjugates experienced negligible hydrolysis after a long-term measurement and exhibited high biostability to the commonly used reducing agents and also in cell lysates, which is in contrast to the conjugates of the maleimide spin label, CT02MA. PD-EPR studies on the FPS1-labeled immunoglobulin G-binding protein (GB1) and second-mitochondrion-derived activator of caspases (Smac) demonstrated that FPS1 enables highly sensitive and long-range distance measurement in the proteins. Importantly, due to its short and rigid aryl linker, the use of FPS1 provides significantly narrower distance distributions than those from the maleimide spin labels CT02MA and OXMA. Moreover, using FPS1, the longest distance measurement (∼6.01 nm) so far in all of the trityl-labeled protein samples has also been achieved in the FPS1-labeled Smac. Overall, this work demonstrates that the SNAr-based aryl groups can be ideally united into the skeleton of trityl radicals, and the resulting spin labels enable sensitive, precise, and ultralong distance measurements in proteins under diverse conditions.