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

Abstract

Integral membrane proteins (IMPs) are pivotal for cellular functions but challenging to investigate. Here, we introduce IC-FPOMP (in-cell fast photochemical oxidation of MPs), a method enabling in situ footprinting of IMPs within live cells. IC-FPOMP generates reactive oxygen radicals from various precursors (TiO2 nanoparticles or H2O2) nearby the membrane. Leveraging a laser and a 96-well plate platform, we achieve high-throughput and rapid footprinting of IMPs. IC-FPOMP of two human IMPs (human glucose transporter-hGLUT1 and human gamma glutamyl carboxylase-hGGCX) were successful, providing footprinting of both the transmembrane and extramembrane regions. Comparative analysis of hGLUT1 in liposomes versus cells shows that the membrane impacts the transporter‘s conformation differently. In-cell drug screening targeting hGLUT1 reveals drug binding behavior in vivo. In summary, IC-FPOMP offers insights on IMP structure-function relationships in cell and facilitates drug discovery.