Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry.
The study of membrane protein structure and enzymology has traditionally been hampered by the inherent insolubility of membrane proteins in aqueous environments and experimental challenges in emulating an in vivo lipid environment. Phospholipid bilayer nanodiscs have recently been shown to be of great use for the study of membrane proteins since they offer a controllable, stable, and monodisperse model membrane with a nativelike lipid bilayer. Here we report the integration of nanodiscs with hydrogen exchange (HX) mass spectrometry (MS) experiments, thereby allowing for analysis of the native conformation of membrane proteins. gamma-Glutamyl carboxylase (GGCX), an approximately 94 kDa transmembrane protein, was inserted into nanodiscs and labeled with deuterium oxide under native conditions. Analytical parameters including sample-handling and chromatographic separation were optimized to measure the incorporation of deuterium into GGCX. Coupling nanodisc technology with HX MS offers an effective approach for investigating the conformation and dynamics of membrane proteins in their native environment and is therefore capable of providing much needed insight into the function of membrane proteins.
Department of Chemistry, Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Christine M
CM
Hebling
Christopher R
CR
Morgan
Darrel W
DW
Stafford
James W
JW
Jorgenson
Kasper D
KD
Rand
John R
JR
Engen
D019735:Carbon-Carbon Ligases / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D017666:Deuterium Oxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D017666:Deuterium Oxide