Membrane proteins constitute approximately one third of all proteins encoded in both prokaryotic and eukaryotic genomes, and are the pharmacological target of many drugs both commercially available and in clinical development.  However, in contrast to their soluble counterparts, high-resolution structures have been determined for only a small fraction of membrane proteins.  Many factors contribute to this disparity, including the conformational flexibility that is inherent and necessary for the relaying of signals and substrates across a lipid bilayer. Thus, biochemical characterization of membrane proteins typically requires detergent-based matrices to facilitate solubility and conformational stabilization, which can complicate traditional strategies for structural characterization such as crystallography and NMR.  At Genentech, we are developing hydroxyl radical footprinting-mass spectrometry (HRF-MS) which utilizes a high power laser to photo-lyse hydrogen peroxide into hydroxyl radicals, initiating the sub-microsecond surface oxidation of solvent exposed amino acid residues.  Subsequent peptide mapping using LC-MS/MS analysis enables the identification and quantification of the oxidized residues corresponding to their solvent accessibility.  When utilized in a comparative analysis, this technique enables identification of protein structural perturbations upon ligand binding. However, the complicated matrices typically required for membrane protein also analysis poses significant challenges to HRF-MS analysis due to sequestering of the hydroxyl radicals. Here we present our strategy for monitoring hydroxyl radical production using a reporter peptide internal standard, and how this strategy was applied to optimize labeling conditions for structural analysis of the essential ATP-binding cassette transport, MsbA.<br/>

Hydroxyl radical footprinting-mass spectrometry (HRF-MS): Probing solvent accessibility changes for the elucidation of membrane-ligand interactions

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.