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

Ming Cheng, Chunyang Guo, Weikai Li, Michael L. Gross

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

A free-radical footprinting approach is described for integral membrane protein (IMP) that extends, significantly, the “fast photochemical oxidation of proteins” (FPOP) platform. This new approach exploits highly hydrophobic perfluoroisopropyl iodide (PFIPI) together with tip sonication to ensure efficient transport into the micelle interior, allowing laser dissociation and footprinting of the transmembrane domains. In contrast to water soluble footprinters, PFIPI 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 VKOR's enzymatic function, and tyrosine iodination does not compromise protease digestion and the subsequent analysis. The locations for the modifications are largely consistent with the corresponding solvent accessibilities, recommending this approach for future membrane protein footprinting.

Original languageEnglish
Pages (from-to)8867-8873
Number of pages7
JournalAngewandte Chemie - International Edition
Volume60
Issue number16
DOIs
StatePublished - Apr 12 2021

Keywords

  • fast photochemical oxidation of proteins
  • iodine radicals
  • membrane proteins
  • micelles
  • protein footprinting

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