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 F1F0-ATP synthase from the extremely thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1. In this study, the genes encoding the F1 moiety of the enzyme complex were cloned from the atp operon into the vector pTrc99A and expressed in Escherichia coli in two variant complexes, F1-wt consisting of subunits α3β3γδε and F1Δδ lacking the entire δ-subunit as a prerequisite for overproduction and crystallization trials. Both F1-wt and F1Δδ were successfully overproduced in E. coli and purified in high yield and purity. F1Δδ was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1 Å 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 F1-wt. Data processing of diffraction patterns showed that F1Δδ crystals belong to the orthorhombic space group P212121 with unit cell parameters of a = 121.70, b = 174.80, and c = 223.50 Å, α, β, γ = 90.000. The asymmetric unit contained one molecule of bacterial F1Δδ with a corresponding volume per protein weight (VM) of 3.25 Å3 Da−1 and a solvent content of 62.1%. Silver staining of single crystals of F1Δδ analyzed by SDS-PAGE revealed four bands α, β, γ, and ε with identical Mr-values as those found in the native F1F0-ATP synthase isolated from strain TA2.A1 membranes. ATPase assays of F1Δδ crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme.