Sun Jie, Saimi Mierxiati, Rempel Don, Cao Qing, Chai Mengqi, Li Weikai, Gross Michael L
Department of Chemistry, Washington University in St. Louis, One Brookings Drive, Box 1134, St. Louis, MO, 63130, USA.
Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, 1311 Cumberland Avenue, Knoxville, TN, 37996-1937, USA.
Angew Chem Int Ed Engl. 2025 May;64(19):e202424779. doi: 10.1002/anie.202424779. Epub 2025 Mar 9.
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 (TiO nanoparticles or HO) 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.
整合膜蛋白（IMPs）对细胞功能至关重要，但研究起来颇具挑战性。本文介绍了IC-FPOMP（细胞内MPs快速光化学氧化），这是一种能够对活细胞内的IMPs进行原位足迹分析的方法。IC-FPOMP从膜附近的各种前体（TiO纳米颗粒或HO）产生活性氧自由基。利用激光和96孔板平台，实现了IMPs的高通量快速足迹分析。两种人类IMPs（人类葡萄糖转运蛋白-hGLUT1和人类γ-谷氨酰羧化酶-hGGCX）的IC-FPOMP分析成功，提供了跨膜区和膜外区的足迹信息。对脂质体和细胞中的hGLUT1进行比较分析表明，膜对转运蛋白构象的影响可能不同。针对hGLUT1的细胞内药物筛选揭示了体内药物结合行为。总之，IC-FPOMP为深入了解细胞中IMPs的结构-功能关系提供了线索，并促进了药物发现。
