In‐Cell Fast Photochemical Oxidation Interrogates the Native Structure of Integral Membrane Proteins

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.