Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.
Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.
复制标题
感觉视紫红质 II/HtrII/Tsr 融合嵌合体的光刺激可在体外激活 CheA 自磷酸化和 CheY 磷酸转移。
DOI：
10.1021/bi034399q
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Spudich,JohnL
中科院分区：
文献类型：
--
作者：
Trivedi,VishwaD;Spudich,JohnL
在大肠杆菌中表达了一种嵌合融合蛋白，该融合蛋白由嗜盐碱单胞菌感觉视紫红质Ⅱ（SR Ⅱ）通过柔性接头与其同源转导蛋白Htr Ⅱ的两个跨膜螺旋融合，随后是Htr Ⅱ近膜胞质片段与大肠杆菌趋化性受体Tsr胞质结构域连接组成。杆菌纯化的融合嵌合体蛋白在脂质体中重组结合E。coliCheA激酶的偶联蛋白CheW的存在下，并激活CheA自磷酸化活性。CheA激酶活性通过融合蛋白的SRII结构域的光激发来刺激，如通过光刺激磷酸转移到E.纯化的体外脂质体系统中的大肠杆菌鞭毛运动反应调节剂CheY。进一步证实了在体外系统的保真度，增加和减少的水平的CheA激活在体外的结果从过度甲基化和甲基化不足的融合蛋白纯化的甲基酯酶和甲基转移酶缺陷E。coli中表达。光激发的甲基化不足的融合蛋白导致在黑暗状态下的磷酸转移增加了3倍。结果直接表明耦合SRII光活化状态组氨酸激酶活性，先前预测的基础上的序列同源性的嗜盐古菌趋光性系统组件的那些ofE。大肠杆菌趋性融合嵌合体提供了体外测量SRII−HtrII分子复合物光信号活性的第一个工具。
A chimeric fusion protein consisting ofNatronomonas pharaonissensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate transducer protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of theEscherichia colichemotaxis receptor Tsr, was expressed inE. coli. Purified fusion chimera protein reconstituted in liposomes binds toE. coliCheA kinase in the presence of the coupling protein CheW, and activates CheA autophosphorylation activity. CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to theE. coliflagellar motor response regulator CheY in the purified in vitro liposomal system. Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion protein purified from methylesterase and methyltransferase-deficientE. coli, respectively. Photoexcitation of the undermethylated fusion protein resulted in a 3-fold increase in phosphotransfer over that of the dark state. The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those ofE. colichemotaxis. The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII−HtrII molecular complexes.