Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy
Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy
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大肠杆菌膜和蛋白脂质体中的寡聚传感器激酶 DcuS：化学交联和 FRET 光谱
DOI：
10.1128/jb.00082-10
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发表时间：
2010
影响因子：
3.2
通讯作者：
W. Erker
中科院分区：
文献类型：
--
作者：
P. Scheu;Yun;J. Bauer;H. Kneuper;T. Basché;G. Unden;W. Erker
摘要DcuS是大肠杆菌DcuSR双组分系统的膜整合传感器组氨酸激酶，对细胞外C4-二羧酸起反应。通过化学交联和荧光共振能量转移（FRET）光谱法，在体外和活细胞中研究了全长DcuS的寡聚状态。通过使用活细胞的无背景光谱的改进方法定量FRET结果，用于确定FRET效率（E）和供体分数{fD =（供体）/[（供体）+（受体）]}。将绿色荧光蛋白的青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）变体与DcuS的功能性融合体用于体内FRET测量。基于非相互作用的膜蛋白和完全相互作用的蛋白（CFP-YFP融合物），定量评价共表达DcuS-CFP和DcuS-YFP的细胞的FRET结果。在活细胞和重组后的纯化重组DcuS的蛋白脂质体，DcuS被发现作为一个二聚体或更高的寡聚体，独立的效应的存在。与二琥珀酰亚胺辛二酸酯的化学交联显示四聚体，除了二聚体，DCuS在蛋白脂质体和细菌的膜，而纯化的DCuS在非变性洗涤剂主要是单体。体内和脂蛋白体中四聚体DcuS的存在和量不依赖于DcuS的浓度。只有膜包埋的DcuS（存在于寡聚状态）是积极的（自动）磷酸化。总体而言，FRET和交联数据表明，在活细胞中，在细菌膜中，并在蛋白脂质体的全长DcuS蛋白在寡聚体状态，包括四聚体的存在。
ABSTRACT DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by f luorescence r esonance e nergy t ransfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.
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