Two-Dimensional Crystallization of Ca-ATPase by Detergent Removal

• Published in: Biophysical journal Vol. 75; no. 3; pp. 1319 - 1329
• Main Authors: Lacapère, Jean-Jacques, Stokes, David L., Olofsson, Anders, Rigaud, Jean-Louis
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.1998
• Subjects: Animals; Biophysical Phenomena; Biophysics; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - isolation & purification; Calcium-Transporting ATPases - ultrastructure; Crystallization; Detergents - isolation & purification; Lipids; Micelles; Microscopy, Electron; Polystyrenes; Protein Conformation; Sarcoplasmic Reticulum - enzymology
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(98)74050-0

By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 Å, b = 54 Å, and γ = 90° for both types of crystals, and projection maps at 15-Å resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca 2+; the presence of ADP gave rise to another type of packing within the unit cell ( a = 86 Å, b = 77 Å, and γ = 90°), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain.