A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase.
A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase.
复制标题
大肠杆菌中的绿色荧光蛋白溶解度筛选揭示了 P 元件转座酶中 GTP 结合结构域的结构域边界。
DOI：
10.1002/pro.499
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Rio,DonaldC
中科院分区：
文献类型：
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作者：
Sabogal,Alex;Rio,DonaldC
三磷酸鸟苷（GTP）的结合和水解事件通常作为蛋白质中的分子开关，调节许多信号分子和运输系统中活性和非活性状态之间的构象变化。黑腹果蝇的P元素转座酶需要GTP结合，在初始位点特异性DNA结合后，沿着其反应途径进行。GTP结合是P元素独有的，可能代表了一种新的转座调节形式，允许结合的转座酶找到第二个位点，环转座DNA进行链切割和切除。GTP结合活性先前已被定位到转座酶蛋白的中心部分；然而，P元件转座酶与已知的GTP结合折叠几乎没有序列一致性。为了鉴定可溶性的、活性的转座酶结构域，采用GFP溶解性筛选法检测随机P元件基因片段inE的溶解性。杆菌。筛选产生了一个在转座酶编码区中心部分已知的GTP结合残基的单克隆。该克隆P元素转座酶中氨基酸275-409为可溶性高表达inE。因此，大肠杆菌具有GTP结合活性，是未来生物化学和结构研究的候选物质。此外，嵌合筛选显示，在P元件转座酶中，一个最小的N端THAP DNA结合域连接到一个扩展的亮氨酸拉链卷曲二聚化域，精确地描绘了初级序列上的DNA结合和二聚化活性。本研究强调了在大型多结构域蛋白上使用基于GFP的溶解度筛选来鉴定高表达的可溶性截断结构域亚区。
Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase ofDrosophila melanogasterrequires GTP binding to proceed along its reaction pathway, following initial site‐specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP‐binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP‐binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments inE. coli. The screen produced a single clone spanning known GTP‐binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed inE.coliand active for GTP‐binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N‐terminal THAP DNA‐binding domain attached to an extended leucine zipper coiled‐coil dimerization domain in the P element transposase, precisely delineating the DNA‐binding and dimerization activities on the primary sequence. This study highlights the use of a GFP‐based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions.