期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 152, 期 2, 页码 140-145出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2005.08.005
关键词
crystallization; X-ray; F-1-ATPase; thermoalkaliphile; 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 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)gamma delta epsilon and F-1 Delta delta lacking the entire delta-subunit as a prerequisite for overproduction and crystallization trials. Both F-1-wt and F-1 Delta delta were successfully overproduced in E. coli and purified in high yield and purity. F-1 Delta delta was crystallized by micro-batch screening yielding three-dimensional crystals that diffracted to a resolution of 3.1 angstrom 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 Delta delta 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 = 223.50 angstrom, alpha, beta, gamma = 90.000. The asymmetric unit contained one molecule of bacterial F-1 Delta delta with a corresponding volume per protein weight (V-M) of 3.25 angstrom(3) Da(-1) and a solvent content of 62.1%. Silver staining of single crystals of F-1 Delta delta analyzed by SDS-PAGE revealed four bands alpha, beta, gamma, and epsilon with identical M-r -values as those found in the native F1F0-ATP synthase isolated from strain TA2A1 membranes. ATPase assays of F-1 Delta delta crystals exhibited latent ATP hydrolytic activity that was highly stimulated by lauryldimethylamine oxide, a hallmark of the native enzyme. (C) 2005 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据