Micelles protect membrane complexes from solution to vacuum.
The ability to maintain interactions between soluble protein subunits in the gas phase of a mass spectrometer gives critical insight into the stoichiometry and interaction networks of protein complexes. Conversely, for membrane protein complexes in micelles, the transition into the gas phase usually leads to the disruption of interactions, particularly between cytoplasmic and membrane subunits, and a mass spectrum dominated by large aggregates of detergent molecules. We show that by applying nanoelectrospray to a micellar solution of a membrane protein complex, the heteromeric adenosine 5'-triphosphate (ATP)-binding cassette transporter BtuC2D2, we can maintain the complex intact in the gas phase of a mass spectrometer. Dissociation of either transmembrane (BtuC) or cytoplasmic (BtuD) subunits uncovers modifications to the transmembrane subunits and cooperative binding of ATP. By protecting a membrane protein complex within a n-dodecyl-beta-d-maltoside micelle, we demonstrated a powerful strategy that will enable the subunit stoichiometry and ligand-binding properties of membrane complexes to be determined directly, by precise determination of the masses of intact complexes and dissociated subunits.
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB21EW, UK.
Nelson P
NP
Barrera
Natalie
N
Di Bartolo
Paula J
PJ
Booth
Carol V
CV
Robinson
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003902:Detergents; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005740:Gases; D005960:Glucosides; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008823:Micelles; D046912:Multiprotein Complexes / Q000737:chemistry; D036103:Nanotechnology; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D014618:Vacuum
D018528:ATP-Binding Cassette Transporters; C050021:BtuC peptide, E coli; C050022:BtuD peptide, E coli; D003902:Detergents; D029968:Escherichia coli Proteins; D005740:Gases; D005960:Glucosides; D008024:Ligands; D008823:Micelles; D046912:Multiprotein Complexes; D021122:Protein Subunits; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate