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Updated: May 24, 2025

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins
Published on: March 11, 2020
In-Cell Fast Photochemical Oxidation Interrogates the Native Structure of Integral Membrane Proteins
Jie Sun1,2, Mierxiati Saimi3, Don Rempel1
1Department of Chemistry, Washington University in St. Louis, One Brookings Drive, Box 1134, St. Louis, MO, 63130, USA.
Abstract:
Integral membrane proteins (IMPs) are pivotal for cellular functions but challenging to investigate. Here, IC-FPOMP (in-cell fast photochemical oxidation of MPs) is introduced, a method enabling in situ footprinting of IMPs within live cells. IC-FPOMP generates reactive oxygen radicals from various precursors (TiO2 nanoparticles or H2O2) near the membrane. Leveraging a laser and a 96-well plate platform, high-throughput and rapid footprinting of IMPs are achieved. IC-FPOMP of two human IMPs (human glucose transporter-hGLUT1 and human gamma-glutamyl carboxylase-hGGCX) are successful, providing footprinting of both the transmembrane and extramembrane regions. Comparative analysis of hGLUT1 in liposomes versus cells shows that the membrane may impact the transporter's conformation differently. In-cell drug screening targeting hGLUT1 reveals drug-binding behavior in vivo. In summary, IC-FPOMP offers insights into IMP structure-function relationships in cells and facilitates drug discovery.
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