Two-Dimensional Crystallization of Ca-ATPase by Detergent Removal

Abstract

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 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A 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 Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), 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.

Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

“…This is similar to the lipid : protein ratios commonly used for Na + K + -ATPase function studies [38]. Our identified nonoxidizing N 2 atmosphere parallels the need because oxidation of tryptophan decreases its fluorescence [30].…”

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

“…Dialysis is the most widely used technique in 2-D crystallization trials. Due to the necessity to scale down the amount of material, microdialysis devices have been used in the form of small compartments (50-100 µl) dialyzed against large solubilized in various detergents and, importantly, some of these 2-D crystals have been useful for high-resolution structural analysis (36,(41)(42)(43).…”

Direct detection of SERCA calcium transport and small-molecule inhibition in giant unilamellar vesicles

“…SUV reconstitution was performed using a mix of two neutrally-charged lipids (80% DOPC + 20% DOPC) that we have shown to maximize SERCA activity [36,37]. In general, reconstitution of SUV proteoliposomes requires a minimum of 100 lipids per protein [38]. Purified SERCA contains ≈ 8 co-purifying lipids (mol/mol) [38], so we typically supplemented purified SERCA with 50 lipids (mol/mol) for storage, and then added an additional 50 lipids (mol/mol) for during detergent-dilution reconstitution, giving a total of ≈ 110 lipid per SERCA in SUV-SERCA.…”

“…In general, reconstitution of SUV proteoliposomes requires a minimum of 100 lipids per protein [38]. Purified SERCA contains ≈ 8 co-purifying lipids (mol/mol) [38], so we typically supplemented purified SERCA with 50 lipids (mol/mol) for storage, and then added an additional 50 lipids (mol/mol) for during detergent-dilution reconstitution, giving a total of ≈ 110 lipid per SERCA in SUV-SERCA. The lipid/protein ratio of 110 was chosen because this condition provides efficient formation of SUV-SERCA with diameter ~0.10–20 µm [39] and because SUV with this diameter range are fusogenic [40].…”