Helical crystallization of two example membrane proteins MsbA and the Ca(2+)-ATPase.
Helical crystallization is a powerful tool for the moderate resolution structure determination of integral membrane proteins, where the insight gained often includes domain architecture and the disposition of α-helical segments. A necessary first step toward helical crystallization involves membrane protein reconstitution, which itself is a powerful technique for structure-function studies of integral membrane proteins. The correct insertion of a detergent-solubilized, purified membrane protein into lipid vesicles (proteoliposomes) can facilitate the functional characterization of the protein in a well-defined, chemically pure environment without interference from other membrane-associated components. In addition, the lipid-to-protein ratio can be controlled during reconstitution to generate a high concentration of a particular membrane protein in the proteoliposomes, which are then suitable for both functional assays and crystallization trials. Traditional approaches to two-dimensional crystallization for electron microscopy rely on dialysis methods for the simultaneous reconstitution and crystallization of a membrane protein [Kühlbrandt, W. (1992). Two-dimensional crystallization of membrane proteins. Q. Rev. Biophys.25, 1-49.], yet some systems allow these two steps to be experimentally separated and independently considered. Some examples of integral membrane proteins that have been reconstituted and crystallized in a helical lattice include cytochrome bc1 complex from bovine heart [Akiba, T., et al. (1996). Three-dimensional structure of bovine cytochrome bc(1) complex by electron cryomicroscopy and helical image reconstruction. Nat. Struct. Biol.3, 553-561.], Escherichia coli melibiose permease [Rigaud, J. L., et al. (1997). Bio-beads: An efficient strategy for two-dimensional crystallization of membrane proteins. J. Struct. Biol.118, 226-235.], a bacterial ATP-binding cassette transporter MsbA [Ward, A., et al. (2009). Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA. J. Struct. Biol.165, 169-175.], and the sarcoplasmic reticulum Ca(2+)-ATPase [Young, H. S., et al. (1997). How to make tubular crystals by reconstitution of detergent-solubilized Ca(2+)-ATPase. Biophys. J.72, 2545-2558.]. The reconstitution and helical crystallization of MsbA and Ca(2+)-ATPase will be the focus of this chapter.
Glaves
John Paul
JP
Department of Biochemistry, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada.
Fisher
Lauren
L
Ward
Andrew
A
Young
Howard S
HS
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003902:Detergents; D008565:Membrane Proteins; C079726:MsbA protein, Bacteria; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D008274:Magnesium