title: Elucidating structural dynamics of integral membrane proteins on native cell surface by hydroxyl radical footprinting and nano LC-MS/MS.
abstract: Although the snapshots of different in vitro conformational states have been intensively studied, current techniques such as nuclear magnetic resonance, X-ray crystallography, and electron microscope method cannot probe the in vivo conformational movements of integral membrane proteins on cell surfaces. Here, we describe a hydroxyl radical protein footprinting coupled to a mass spectrometry detection technique to probe the structural dynamics of a membrane protein directly on the native cell surface. This method uses in situ generation of hydroxyl radicals to oxidize and covalently modify integral membrane proteins on the cell surface. To explain this technique in detail, we use the porin OmpF as an example, although the method may be applied to study any membrane protein. Footprinting results show that the surface mapping data of OmpF are consistent with its current crystallographic structure. In addition, this technique also enables the detection of in vivo voltage gating of porin OmpF for the first time. This novel cell surface footprinting method coupled with MS analysis can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhu
authors.forename: Tiannan
authors.initials: T
authors.lastname: Guo
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
mesh_terms: D000595:Amino Acid Sequence; D002470:Cell Survival; D019295:Computational Biology; D048168:Escherichia coli K12 / Q000166:cytology; D017665:Hydroxyl Radical / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D012997:Solvents / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D012997:Solvents; D017665:Hydroxyl Radical