title: Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.
abstract: Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Juho
authors.initials: J
authors.lastname: Kellosalo
authors.forename: Tommi
authors.initials: T
authors.lastname: Kajander
authors.forename: Riina
authors.initials: R
authors.lastname: Honkanen
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
mesh_terms: D003460:Crystallization; D003902:Detergents / Q000737:chemistry; D004789:Enzyme Activation; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D041543:Pyrobaculum / Q000201:enzymology / Q000235:genetics; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics; D014961:X-Ray Diffraction
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011755:Pyrophosphatases