Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

Abstract

Afree-radical footprinting approach is described for integral membrane protein (IMP) that extends,s ignificantly, the "fast photochemical oxidation of proteins" (FPOP) platform. This new approache xploits highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication to ensure efficient transport into the micelle interior,a llowing laser dissociation and footprinting of the transmembrane domains.I nc ontrast to water soluble footprinters,P FIPI footprints both the hydrophobic intramembrane and the hydrophilic extramembrane domains of the IMP vitamin K epoxide reductase (VKOR). The footprinting is fast, giving high coverage for Tyr( 100 %) and Trp. The incorporation of the reagent with sonication does not significantly affect VKORse nzymatic function, and tyrosine iodination does not compromise protease digestion and the subsequent analysis.T he locations for the modifications are largely consistent with the corresponding solvent accessibilities,r ecommending this approachf or future membrane protein footprinting.