Perraud A L, Rippe K, Bantscheff M, Glocker M, Lucassen M, Jung K, Sebald W, Weiss V, Gross R
Lehrstuhl für Mikrobiologie, Biozentrum, Universität Würzburg, Germany.
Biochim Biophys Acta. 2000 May 23;1478(2):341-54. doi: 10.1016/s0167-4838(00)00052-2.
Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).
对源自大肠杆菌和百日咳博德特氏菌非传统的EvgAS和BvgAS双组分磷酸化信号转导系统的信号蛋白或结构域的生物物理和生化特性进行了研究。通过非变性凝胶电泳、凝胶渗透实验和分析超速离心对效应蛋白EvgA和BvgA以及截短的EvgS和BvgS衍生的信号蛋白（包含接收结构域和含组氨酸的磷酸转移（HPt）结构域或仅包含HPt结构域）的寡聚化进行了表征。通过不同方法获得的结果与非磷酸化的EvgA和BvgA蛋白在溶液中为二聚体且解离常数显著低于1微摩尔相一致。相反，观察到EvgS和BvgS的所有传感器衍生结构域在体外均为单体。未检测到磷酸化诱导的C端组氨酸激酶结构域寡聚化刺激的迹象。与这些数据一致，表面等离子体共振研究显示EvgA与固定化的EvgS HPt结构域相互作用的化学计量比为2:1，亲和常数为1.24×10⁶ M⁻¹。
