Optimizing oriented planar-supported lipid samples for solid-state protein NMR.
Sample orientation relative to the static magnetic field of an NMR spectrometer allows study of membrane proteins in the lipid bilayer setting. The straightforward preparation and handling of extremely thin mica substrates with consistent surface properties has prompted us to examine oriented phospholipid bilayer and hexagonal phases on mica. The spectral characteristics of oriented lipid samples formed on mica are as good as or better than those on glass. Nine solvents with varying dielectric constants were used to cast lipid films or for vesicle spreading; film characteristics were then compared, and static solid-state 31P-NMR was used to characterize the degree of orientation of the hydrated lipid species. Lipids with four headgroup chemistries were tested: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Solvent affected orientation of POPG, DOPA, and DOPE, but not POPC. Film characteristics varied with solvent, with ramifications for producing homogeneous oriented lipid samples. POPC was used to optimize the amount of lipid per substrate and compare hydration methods. POPG did not orient reproducibly, whereas POPG-POPC mixtures did. DOPA showed 1-2 oriented states depending upon hydration level and deposition method. DOPE formed an oriented hexagonal phase that underwent a reversible temperature-induced phase transition to the oriented bilayer phase.
Rainey
Jan K
JK
Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta, Canada.
Sykes
Brian D
BD
D000538:Aluminum Silicates / Q000737:chemistry*; D016880:Anisotropy; D008051:Lipid Bilayers / Q000032:analysis / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D008567:Membranes, Artificial; D044367:Phase Transition; D012997:Solvents / Q000737:chemistry; D013048:Specimen Handling / Q000379:methods*
D000538:Aluminum Silicates; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D012997:Solvents; C011934:mica