How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.
In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.
Young
H S
HS
Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York 10016, USA.
Rigaud
J L
JL
Lacapère
J J
JJ
Reddy
L G
LG
Stokes
D L
DL
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D003460:Crystallization; D003902:Detergents; D066298:In Vitro Techniques; D008055:Lipids / Q000032:analysis; D008854:Microscopy, Electron; D011506:Proteins / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D012995:Solubility
D003902:Detergents; D008055:Lipids; D011506:Proteins; D000252:Calcium-Transporting ATPases