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首页> 外文期刊>Journal of Molecular Biology >Nucleotide Dependent Monomer/Dimer Equilibrium of OpuAA, the Nucleotide-binding Protein of the Osmotically Regulated ABC Transporter OpuA from Bacillus subtilis.
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Nucleotide Dependent Monomer/Dimer Equilibrium of OpuAA, the Nucleotide-binding Protein of the Osmotically Regulated ABC Transporter OpuA from Bacillus subtilis.

机译：OpuAA的核苷酸依赖性单体/二聚体平衡，OpsAA是渗透调节的来自枯草芽孢杆菌的ABC转运蛋白OpuA的核苷酸结合蛋白。

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摘要

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.
机译：枯草芽孢杆菌的OpuA系统是依赖底物结合蛋白的ABC转运蛋白超家族的成员，并在高渗生长条件下用于吸收相容的溶质甘氨酸甜菜碱。在这里，我们表征了枯草芽孢杆菌OpuA转运蛋白的核苷酸结合蛋白（OpuAA）。 OpuAA作为六组氨酸标签融合蛋白在大肠杆菌中异源表达，并通过亲和力和尺寸排阻色谱（SEC）纯化至均一。观察到OpuAA的动态单体/二聚体平衡，并且K（D）值确定为6 microM。在高离子强度测定条件下，单体/二聚体的相互转化减少，这使得可以通过SEC分离两种物质，并对单体和二聚体OpuAA进行单独分析。在存在1 M NaCl的情况下，单体OpuAA显示出基础ATPase活性（K（M）= 0.45 mM; k（2）= 2.3 min（-1）），而二聚体OpuAA在此条件下显示出很少的ATPase活性。核苷酸的添加影响了OpuAA的单体/二聚体比率，表明了其催化周期中的不同寡聚状态。在水解后的条件下（例如ADP / Mg（2+）），单体是优选的种类，而二聚体主导了无核苷酸和ATP结合的状态。单体或二聚体OpuAA / ATP复合物的亲和力和化学计量通过荧光ATP类似物TNP-ATP确定。一分子TNP-ATP以单体状态结合，并且在OpuAA的二聚体状态中检测到两个TNP-ATP分子。 TNP-ADP / Mg（2+）与二聚体OpuAA的结合诱导构象变化，导致二聚体的衰变。根据我们的数据，我们提出了一个模型，该模型将OpuAA的低聚状态的变化与ATP水解耦合在一起。

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