Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane‐Spanning Protein

Abstract

Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study a carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane-spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein-protein interactions between the trimer subunits remained unlabelled, thus revealing their location.

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…Oldham and co‐workers compared TPDs to PL for several different proteins and showed that TPDs exhibit a higher reaction efficiency than PL. Given the amphipathic nature of the TPDs, Oldham, Moses, and co‐workers successfully demonstrated that this reagent can label the hydrophobic transmembrane domains of a membrane protein.…”

Fluorierte Reagenzien in der Strukturproteomik

“…Oldham und Mitarbeiter verglichen TPDs mit PL in Kombination mit verschiedenen Proteinen und zeigten, dass TPDs effizienter reagieren als PL . Robinson, Oldham und Mitarbeiter zeigten, dass FPDs dank ihrer amphiphatischen Eigenschaften hydrophobe Transmembrandomänen eines Membranproteins markieren können.…”

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments

“…glutamine and histidine). Other radicala nd non-radical chemical labelling strategies have also been used to report on membrane protein structure through specific or promiscuous amino acid labelling,f or example, using carbene footprinting, [5] diethylpyrocarbonate (DEPC) labelling, [6] and cysteines pecific N-ethylmaleimide (NEM) and lysine specific succinic anhydride labelling. [7] Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) HDX-MS is at ype of footprintingt echnique which measures the extent and rate of exchange of protein backbone amide hydrogens for deuterium.…”

“…glutamine and histidine). Other radical and non‐radical chemical labelling strategies have also been used to report on membrane protein structure through specific or promiscuous amino acid labelling, for example, using carbene footprinting, diethylpyrocarbonate (DEPC) labelling, and cysteine specific N ‐ethylmaleimide (NEM) and lysine specific succinic anhydride labelling …”