Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer.
Two-dimensional crystals of a membrane protein, the proton ATPase from plant plasma membranes, have been obtained by a new strategy based on the use of functionalized, fluorinated lipids spread at the air-water interface. Monolayers of the fluorinated lipids are stable even in the presence of high concentrations of various detergents as was established by ellipsometry measurements. A nickel functionalized fluorinated lipid was spread into a monolayer at the air-water interface. The overexpressed His-tagged ATPase solubilized by detergents was added to the subphase. 2D crystals of the membrane protein, embedded in a lipid bilayer, formed as the detergent was removed by adsorption. Electron microscopy indicated that the 2D crystals were single layers with dimensions of 10 microm or more. Image processing yielded a projection map at 9 A resolution, showing three well-separated domains of the membrane-embedded proton ATPase.
Lebeau
L
L
Laboratoire de Synthèse Bioorganique associé au CNRS Université Louis Pasteur, Illkirch, 67401, France. lebeau@aspirine.u.strasbg.fr
Lach
F
F
Vénien-Bryan
C
C
Renault
A
A
Dietrich
J
J
Jahn
T
T
Palmgren
M G
MG
Kühlbrandt
W
W
Mioskowski
C
C
D000327:Adsorption; D000388:Air; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology; D002614:Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D020285:Cryoelectron Microscopy; D003460:Crystallization; D003902:Detergents / Q000378:metabolism* / Q000494:pharmacology; D005461:Fluorine / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000138:chemical synthesis / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008567:Membranes, Artificial; D008823:Micelles; D009532:Nickel / Q000037:antagonists & inhibitors / Q000378:metabolism; D011312:Pressure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D012995:Solubility / Q000187:drug effects; D014867:Water / Q000378:metabolism
D002614:Chelating Agents; D003902:Detergents; D008051:Lipid Bilayers; D008055:Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D008823:Micelles; D011993:Recombinant Fusion Proteins; D014867:Water; D005461:Fluorine; D009532:Nickel; D006180:Proton-Translocating ATPases