Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus.
Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus.
复制标题
鉴定嗜热脂肪芽孢杆菌 SbsB S 层蛋白中的组装抑制和组装耐受位点。
DOI：
10.1016/j.jmb.2009.10.012
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发表时间：
2010
影响因子：
5.6
通讯作者：
Kinns H
中科院分区：
文献类型：
--
作者：
Kinns H
表面层（S 层）蛋白质自组装成二维晶格，覆盖所有古细菌和许多细菌的细胞壁。我们已经生成了高溶解度的组装阴性蛋白质变体，这将有助于高分辨率结构测定。使用插入诱变筛选获得来自嗜热脂肪芽孢杆菌 PV72/p2 的 S 层蛋白 SbsB 的组装阴性版本。血凝素表位标签被插入到先前半胱氨酸可及性筛选已知位于单体蛋白表面的 23 个氨基酸位置处。使用有限的蛋白水解、圆二色性和荧光来探测表位插入是否影响单体的二级和三级结构，同时使用电子显微镜和尺寸排阻色谱来检查蛋白质的自组装能力。该筛选不仅鉴定了具有天然折叠的组装受损突变体，而且还产生了正确折叠的自组装突变体，适合展示用于生物医学和生物物理应用以及冷冻电子显微镜成像的表位。我们的研究标志着 S 层结构分析迈出了重要一步。此外，协同插入和半胱氨酸诱变方法可能适用于其他超分子组装体。
Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies.
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DOI：
10.1111/j.1365-2818.2003.01270.x
发表时间：
2003-12-01
期刊：
JOURNAL OF MICROSCOPY-OXFORD
影响因子：
--
作者：
Györvary, ES;Stein, O;Sleytr, UB
通讯作者：
Sleytr, UB
影响因子：
6.8
作者：
Lanman, J;Prevelige, PE
通讯作者：
Prevelige, PE
影响因子：
4.8
作者：
Howorka, S;Sára, M;Bayley, H
通讯作者：
Bayley, H
影响因子：
4.8
作者：
Carreira, A;Menéndez, M;Mateu, MG
通讯作者：
Mateu, MG
影响因子：
3.2
作者：
S. Hovmöller
通讯作者：
S. Hovmöller