PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding
PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding
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DOI：
10.1128/jb.186.20.6782-6791.2004
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发表时间：
2004-10-01
影响因子：
3.2
通讯作者：
Taylor, BL
中科院分区：
文献类型：
--
作者：
Herrmann, S;Ma, QH;Taylor, BL
大肠杆菌中的Aer蛋白是一种膜结合的、含有FAD的趋氧性和能量传感器，其可监测电子传递系统的氧化还原状态。FAD与Aer的结合需要N-末端PAS结构域和F1区中的残基以及C-末端RAMP结构域。其他PAS蛋白的PAS结构域可溶于水。为了研究PAS结构域的性质，我们从大肠杆菌中亚克隆了aer基因的片段。将编码PAS结构域的两种PAS结构域分别与His标签和His标签连接，并在大肠杆菌中表达。杆菌通过凝胶过滤层析将20-kDa His（6）-Aer（2-166）PAS-F1片段纯化为800-kDa复合物，并通过N-末端测序将相关蛋白鉴定为伴侣蛋白GroEL。在可溶性级分中发现的Aer的N-末端片段中没有一个从GroEL中释放，这表明这些肽在水性环境中不能正确折叠，并且需要PAS结构域外部的基序进行正确折叠。与该模型一致，包括膜结合区和部分（Aer（2-231））或全部（Aer（2-285））HAMP结构域的肽片段插入膜中，表明它们被GroEL释放。Aer（2-285）而非Aer（2-231）结合FAD，证实了在稳定FAD结合中需要RAMP结构域。这些结果提出了一个有趣的可能性，即FAD结合所需的PAS结构域以外的残基是形成PAS天然折叠所必需的。
The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal RAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His, tags and expressed the PAS peptides in E. coli. The 20-kDa His(6)-Aer(2-166) PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer(2-231)) or all (Aer(2-285)) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer(2-285), but not Aer(2-231), bound FAD, confirming the requirement for the RAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.