Abstract

Mass spectrometry-based footprinting can probe higher order structure of soluble proteins in their native states and serve as a complement to high-resolution approaches. Traditional footprinting approaches, however, are hampered for integral membrane proteins because their transmembrane regions are not accessible to solvent, and they contain hydrophobic residues that are generally unreactive with most chemical reagents. To address this limitation, we bond photocatalytic titanium dioxide (TiO2) nanoparticles to a lipid bilayer. Upon laser irradiation, the nanoparticles produce local concentrations of radicals that penetrate the lipid layer, which is made permeable by a simultaneous laser-initiated Paternò–Büchi reaction. This approach achieves footprinting for integral membrane proteins in liposomes, helps locate both ligand-binding residues in a transporter and ligand-induced conformational changes, and reveals structural aspects of proteins at the flexible unbound state. Overall, this approach proves effective in intramembrane footprinting and forges a connection between material science and biology.

Highlights

  • Mass spectrometry-based footprinting can probe higher order structure of soluble proteins in their native states and serve as a complement to high-resolution approaches

  • The likely reactive species in TiO2 photocatalysis are the holes on the TiO2 surface, hydroxyl radicals, superoxide ions, and singlet oxygen, in addition to trace amounts of H2O2 and O2

  • In NanoPOMP, hydroxyl radicals likely play the major role in initiating the footprinting

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Summary

Introduction

Mass spectrometry-based footprinting can probe higher order structure of soluble proteins in their native states and serve as a complement to high-resolution approaches. This strategy, named NanoPOMP (NP-promoted photochemical oxidation of membrane proteins), yields high coverage in the TM region of bacterial vitamin K epoxide reductase (VKOR) and enables footprinting the conformational changes of human glucose transporter 1 (hGLUT1).

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