Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.
Recently, we reported the cloning of the atp operon encoding for the F(1)F(0)-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F(1) moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F(1)-wt consisting of subunits alpha(3)beta(3)gammadeltaepsilon and F(1)Deltadelta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F(1)-wt and F(1)Deltadelta were successfully overproduced in E. coli and purified in high yield and purity. F(1)Deltadelta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1A using a synchrotron radiation source. After establishing cryo and dehydrating conditions, a complete set of diffraction data was collected from a single crystal. No crystals were obtained with F(1)-wt. Data processing of diffraction patterns showed that F(1)Deltadelta crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell parameters of a=121.70, b=174.80, and c=223.50A, alpha, beta, gamma=90.000. The asymmetric unit contained one molecule of bacterial F(1)Deltadelta with a corresponding volume per protein weight (V(M)) of 3.25A(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F(1)Deltadelta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M(r)-values as those found in the native F(1)F(0)-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F(1)Deltadelta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.
Stocker
Achim
A
Institute of Microbiology ETH Zürich, ETH Hönggerberg, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland.
Keis
Stefanie
S
Cook
Gregory M
GM
Dimroth
Peter
P
D001407:Bacillus / Q000737:chemistry* / Q000235:genetics* / Q000254:growth & development; D002384:Catalysis; D003080:Cold Temperature; D003460:Crystallization; D018360:Crystallography, X-Ray; D003902:Detergents / Q000494:pharmacology; D004123:Dimethylamines / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016622:Silver Staining; D017356:Synchrotrons; D014961:X-Ray Diffraction
D003902:Detergents; D004123:Dimethylamines; D021122:Protein Subunits; D011994:Recombinant Proteins; C014518:dodecyldimethylamine oxide; D006180:Proton-Translocating ATPases