TY - JOUR
T1 - Free-Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication
AU - Cheng, Ming
AU - Guo, Chunyang
AU - Li, Weikai
AU - Gross, Michael L.
N1 - Publisher Copyright:
© 2021 Wiley-VCH GmbH
PY - 2021/4/12
Y1 - 2021/4/12
N2 - 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.
AB - 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.
KW - fast photochemical oxidation of proteins
KW - iodine radicals
KW - membrane proteins
KW - micelles
KW - protein footprinting
UR - http://www.scopus.com/inward/record.url?scp=85102292225&partnerID=8YFLogxK
U2 - 10.1002/anie.202014096
DO - 10.1002/anie.202014096
M3 - Article
C2 - 33751812
AN - SCOPUS:85102292225
SN - 1433-7851
VL - 60
SP - 8867
EP - 8873
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
IS - 16
ER -